MSTO1 encodes a cytosolic mitochondrial fusion protein, misato homolog 1 or MSTO1. While the full genotype–phenotype spectrum remains to be explored, pathogenic variants in MSTO1 have recently been reported in a small number of patients presenting with a phenotype of cerebellar ataxia, congenital muscle involvement with histologic findings ranging from myopathic to dystrophic and pigmentary retinopathy. The proposed underlying pathogenic mechanism of MSTO1-related disease is suggestive of impaired mitochondrial fusion secondary to a loss of function of MSTO1. Disorders of mitochondrial fusion and fission have been shown to also lead to mitochondrial DNA (mtDNA) depletion, linking them to the mtDNA depletion syndromes, a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of cellular mtDNA content. However, the consequences of pathogenic variants in MSTO1 on mtDNA maintenance remain poorly understood. We present extensive phenotypic and genetic data from 12 independent families, including 15 new patients harbouring a broad array of bi-allelic MSTO1 pathogenic variants, and we provide functional characterization from seven MSTO1-related disease patient fibroblasts. Bi-allelic loss-of-function variants in MSTO1 manifest clinically with a remarkably consistent phenotype of childhood-onset muscular dystrophy, corticospinal tract dysfunction and early-onset non-progressive cerebellar atrophy. MSTO1 protein was not detectable in the cultured fibroblasts of all seven patients evaluated, suggesting that pathogenic variants result in a loss of protein expression and/or affect protein stability. Consistent with impaired mitochondrial fusion, mitochondrial networks in fibroblasts were found to be fragmented. Furthermore, all fibroblasts were found to have depletion of mtDNA ranging from 30 to 70% along with alterations to mtDNA nucleoids. Our data corroborate the role of MSTO1 as a mitochondrial fusion protein and highlight a previously unrecognized link to mtDNA regulation. As impaired mitochondrial fusion is a recognized cause of mtDNA depletion syndromes, this novel link to mtDNA depletion in patient fibroblasts suggests that MSTO1-deficiency should also be considered a mtDNA depletion syndrome. Thus, we provide mechanistic insight into the disease pathogenesis associated with MSTO1 mutations and further define the clinical spectrum and the natural history of MSTO1-related disease.The online version of this article (10.1007/s00401-019-02059-z) contains supplementary material, which is available to authorized users. Mitochondria maintain and express their own genome (mtDNA), typically present in 100–1000 copies per cell and organized into nucleoprotein structures known as nucleoids , 18. TheMtDNA depletion syndromes are a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of mitochondrial genomes , 33. Of MFN2 and OPA1 cause severe mtDNA depletion and multiple mtDNA deletions  of the four had evidence of myotonia on EMG, and complex repetitive discharges were present in P8 . Lower extremity muscle MRI imaging was available for four patients and revealed a spectrum of involvement ranging from severe involvement of the muscles of the upper leg with apparent fatty replacement of all muscles except for the semimembranosus and the biceps femoris ; p.(Phe217Leu)) to mild fatty infiltration of upper and lower leg muscles   in 15 patients from 12 independent families consistent with the bi-allelic recessive mode of inheritance , we identified apparent homozygous or compound heterozygous variants in MSTO1 pathogenic variant in P7. Targeted sequencing confirmed that this variant was paternally inherited, while the mother was found to be negative for the variant. Subsequent testing using exon-level oligo CGH array identified a presumed maternally inherited deletion in P7 encompassing at least exons 9-14 of the MSTO1 gene and extending to include both the YY1AP1 as well as the DAP3 genes (Genomic Coordinates: arr[GRCh37] 1q22(155582110_155708204)x1). Recessive pathogenic variants in YY1AP1 have been reported in association with Grange syndrome (OMIM 607860). DAP3 has not yet been associated with human disease.Trio WES identified an apparently homozygous p.(Leu450Phe) MSTO1 variant inherited from a clinically unaffected father. Whole genome sequencing (WGS) in P9 confirmed this heterozygous missense variant but did not identify a second MSTO1 allele in compound heterozygosity. Exon-level oligo CGH testing for P9 was normal. RNA sequencing (RNA-seq) analysis to identify any possible transcriptional aberrations in MSTO1 for P9 was inconclusive, and no splice aberrations were identified in P9. Attempts to evaluate allele balance at the hg19: chr1:155583319 variant via RNA-seq were unsuccessful due to insufficient coverage in the region, likely due to the presence of a highly homologous pseudogene.Trio WES for P9 identified a heterozygous p.(Tyr478Cys) MSTO1 variants was consistent with bi-allelic recessive inheritance, except for P9 (p.(Tyr478Cys); missing) in whom the presumed maternally inherited allele has not yet been identified, and for P13 (p.(Phe217Leu); p.(Phe217Leu)) in whom parental DNA was not available. Variants identified were predicted to be damaging and either absent or extremely rare  in Genome Aggregation Database (GnomAD) and Exome Aggregation Consortium (ExAC) except for the p.(Arg279His) variant. This particular variant was listed with an allele frequency of 0.00019 in ExAC and 0.00026 in GnomAD with one reported homozygous individual , it is intriguing that all patient fibroblasts containing combinations of missense mutations exhibit a similar cellular phenotype. It is also worth noting that MSTO1 variants do not have gross effects on the expression of mitochondrial fusion proteins (MFN1/2 and OPA1)  Fig. b.Fig. 3PMSTO1 patient fibroblasts. Consistent with previous reports, visualized mitochondrial networks in patient fibroblasts were fragmented compared to control fibroblasts  ; p.(Arg279Cys)].The delayed motor development seen in our patients could be due to the cerebellum volume loss, the muscular dystrophy or more likely a combination of both. Review of brain MRI imaging in our cohort demonstrated that the decrease in cerebellar volume is evident in both hemispheres; the volume loss in the vermis is present at a very young age (as observed on the first MRIs obtained) and is non-progressive on follow-up imaging in the majority of patients. Hence, it is possible that the cerebellar volume loss reflects more of a hypoplasia rather than an early-onset progressive atrophy, or a combination thereof. In early cerebellar hypoplasias that interfere with normal cerebellar development, granule cell proliferation deficiency as well as some granular cell loss may underlie an overall smaller cerebellar cortical volume . This maISPD and GMPPB may manifest with phenotypes reminiscent of recessive MSTO1-related disease , WES identified an apparently homozygous MSTO1 missense variant in the absence of consanguinity; however, subsequent WGS and RNA sequencing failed to identify the deletion due to the inherent difficulties of mapping highly homologous regions. Targeted array CGH analysis with exon-level resolution was able to identify the exon 9-14 deletion. Given the duplicated and thus highly similar regions in this analysis, there were only three probes discriminating MSTO1 from MSTO2P; therefore, the mapping of the deletions to MSTO1 is still ambiguous. Given the diagnostic confidence in the disease phenotype, the reduced MSTO1 protein in this patient’s fibroblasts and the previously identified rare, predicted to be damaging missense MSTO1 variant, we suspect that the deletion is likely encompassing MSTO1. In contrast, in patient P9 (p.(Tyr478Cys); missing), who also presented with the disease phenotype and reduced MSTO1 protein levels, extensive next-generation based-sequencing including array CGH analysis only yielded a single heterozygous rare, predicted to be damaging missense MSTO1 variant. A pathogenic variant on the other allele was not readily detectable with available technology, suggesting a more complex genomic re-arrangement which may be copy number neutral. Validation work is in progress; however, this work is significantly complicated by the high sequence similarity between the duplicated genomic regions of the locus. Thus proper diagnosis of MSTO1-deficiency may require specialized sequencing strategies, triggered by proper phenotypic recognition through detailed clinical examination, brain MRI and if needed MSTO1 protein analysis in fibroblasts [The  lineage , which aectively . This hiroblasts .MSTO1 fibroblast lines characterized, MSTO1 protein was reduced , further suggests that impairments in mitochondrial dynamics are upstream of mtDNA depletion in MSTO1 patient fibroblasts. Nonetheless, the exact link between mtDNA copy number and enlarged nucleoids remains unresolved.While the mechanistic link between mitochondrial fusion/fission dynamics and loss of mtDNA content remains unresolved, the present study adds another mitochondrial fusion protein, MSTO1 to the list of mitochondrial dynamics proteins that are implicated in the maintenance of mtDNA content. Our observation of enlarged nucleoids using PicoGreen staining is of interest in this context. The enlarged nucleoids could reflect changes in the topology of mtDNA that affect the ability of the dye to intercalate . Alternasts Fig. a. Meanwhi.e. nucleoid clumping), in conjunction with reduced mitochondrial fusion, impairs mtDNA maintenance leading to mtDNA depletion.It is also notable that mitochondrial fission was recently shown to be important for mtDNA nucleoid segregation following mtDNA replication  and thatMSTO1 patient fibroblasts  is consistent with the levels of mtDNA depletion reported in fibroblasts from OPA1 or MFN2 patients [MSTO1-related disease reported within our cohort is in contrast to the frequently highly variable clinical spectrum observed in mitochondrial diseases with childhood onset due to either nuclear DNA or mtDNA pathogenic variants. Thus, the consequences of abnormal mitochondrial dynamics caused by recessive MSTO1 pathogenic variants may be less susceptible to acutely variable changes in metabolic demands, while likely incorporating a developmental component given the early clinical manifestations and neuroimaging abnormalities. This consistent phenotype of MSTO1-deficiency reported to date also seems in contrast to the single family with a dominantly acting MSTO1 variant in whom psychiatric manifestations including schizophrenia and autism were leading clinical features, while muscle weakness was mild and cerebellar involvement was not reported [MSTO1 investigated to date seem to impair protein stability in fibroblasts, which is consistent with a loss of function mechanism, while not providing further insight into functional subdomains of the protein. Since haploinsufficiency, as is present in some of the heterozygous parents in our cohort, does not cause a clinical phenotype, the mechanism for the dominantly acting heterozygous mutation may be dominant-negative in nature with perhaps different physiological consequences [Importantly, the degree of mtDNA depletion we see in patients , 46, 49 patients . The clireported , thus suequences .MSTO1 result in fragmented mitochondrial networks with mtDNA depletion and nucleoid abnormalities highlight a previously unappreciated role for MSTO1 in the maintenance of the mitochondrial genome. Taken together, our findings newly characterize MSTO1-deficiency as a syndrome of both impaired mitochondrial fusion as well as of mtDNA depletion, while clinically manifesting with a remarkably consistent phenotype.Despite evidence for the role of MSTO1 in mitochondrial fusion, the molecular structure of the MSTO1 protein and the exact mechanism by which MSTO1 performs this function directly or via mediators are still unknown. Our findings that bi-allelic loss-of-function variants in Supplementary material 1 (PDF 153 kb)Below is the link to the electronic supplementary material.


Comparison of mitochondrial DNA (mtDNA) methylation patterns in oocytes, blastocysts and ovarian granulosa cells indicates hitherto unsuspected dynamics. Oocytes and blastocysts recovered from cows subjected to ovarian stimulation and from non-stimulated abattoir ovaries were analyzed using bisulphite transformation of DNA followed by whole genome sequencing. The cow is a recognized as a good model for human oocyte and pre-implantation development. The number of mtDNA copies is high in oocytes  and early embryos, resulting in very high coverage (>3000x) and very low p values for each of 716 cytosine-based nucleosides. Methylation ratio was lowest in oocytes, following by blastocysts then granulosa cells and was not restricted to CG sites but was found also at CHG and CHH sites. The initial methylation pattern is conserved during the first week of life but not in somatic cells. RNA analysis of mitochondria encoded genes showed a significant inverse correlation between methylation and expression for almost all sequences. Methylation was more extensive in somatic tissues from mature animals than in immature pre-pubertal animals. Our findings suggest that mtDNA methylation might play a programming role during gametogenesis and would be subject to epigenetic regulation according to environment and/or maternal maturity. They are present in all tissues, including in oocytes where they go through a selection bottleneck to ensure the passage of the most functional organelles to the next generation2. It should be kept in mind that mitochondria are not transmitted via spermatozoa to the offspring3. The role of mitochondria in mammalian oocytes is complex and changes as the organelles acquire a unique morphology associated with a certain level of quiescence4. Several studies indicate that mitochondrial function is important both in peri-fertilization events in oocytes and in post-fertilization events in early embryos5. A peculiar feature of mitochondria is possession of a genome that encodes only a few of the thousands of genes required for their function. In the case of mammalian cells, mitochondrial DNA (mtDNA) encodes 13 protein components of the oxidative phosphorylation system , as well as mitochondria-specific small and large ribosomal RNA fragments and an array of 22 transfer RNA molecules6. Continuous crosstalk between mitochondria and the nucleus is necessary to ensure that both organelles mobilize the right components for proper function. Mitochondrial genes in the nucleus are under the same type of regulation as non-mitochondrial genes, in which transcription factors, histones and the chromatin environment determine the level of expression of mRNA and ensure its transfer to the cytoplasm for translation, as well as incorporation of the resulting protein into organelles7. On the other hand, the structure of the mitochondrial genome is quite different, consisting of a single 16 kb circular molecule that lacks nucleosomes or histones but rather self-organizes into structures called nucleoids8. The genes are positioned in a polycistronic sequence, and transcription as well as replication begins in the region called the D-Loop, in which the regulatory sequences are located without exons9. Since mitochondrial DNA is not part of the reference system used to analyse sequencing data, these genes are rarely included in microarrays, and therefore, little is known about their expression10. In reproductive tissues, they are rarely studied except in association with specific mitochondrial dysfunctions or mutations in humans11.Mitochondria play an important role in all animal cells as a metabolic energy provider, as well as a regulator of many cellular processes including apoptosis or cell suicide12. Such defects can be induced by several metabolic conditions and are believed to participate in the transmission of F1 phenotypes associated with mitochondrial dysfunction13. However, it is not known if these changes are associated with the nuclear or the mitochondrial genome. It is known that metabolic disorders in many mammalian species can be transmitted through epigenetic inheritance via the oocyte or the sperm cell14. Although it is technically difficult to measure DNA methylation in individual oocytes16, the information obtained so far indicates that epigenetic components (histones and DNA methylation) may play a role in metabolic programming of the future individual17. It has been shown that babies conceived via assisted reproductive technologies may display some phenotypical alterations , however, it is expected that the cardiovascular and metabolic risk factors found in childhood and tracking into adulthood could be worse in later life, and may be responsible for chronic cardiometabolic disease18. This leads to the important question whether maternal metabolic information is passed on to offspring via epigenetic modification of the mitochondrial genome.A related area of study on oocyte mitochondria concerns the transmission of defective organelles in specific metabolic conditions. For example, female mice fed a high-fat diet display modified mitochondrial morphology and function19. Although not completely characterized, evidence is mounting that methylation plays a role in mtDNA gene expression and replication21. Furthermore, it appears that any cytosine nucleoside within mtDNA can be methylated, and is not restricted to CG dinucleotides22. This non-animal-cell characteristic23 also fits the recent discovery that oocytes bear substantially more non-CG methylation than somatic cells24. These new lines of evidence suggest that gene expression in oocytes and their mitochondria might be subject to a new context of DNA methylation either for an immediate or prolonged effect following fertilization and beyond. The aim of this study was to substantiate and explore the potential role of maternal mtDNA methylation in mitochondria on gene expression by comparing oocytes, blastocysts and granulosa cell mtDNA methylation using whole-genome bisulphite sequencing to determine (1) the effects of different ovarian environments on germinal vesicle oocyte (GVO) mtDNA methylation, (2) the developmental effects of these different ovarian environments on blastocyst mtDNA methylation, (3) the link between mtDNA methylation level and gene expression in oocytes and blastocysts, and (4) the effects of female maturity on granulosa cells mtDNA methylation. The results indicate that bovine mitochondrial DNA shows a unique signature in oocytes and blastocysts compared to the associated somatic (granulosa) cells; an inverted methylation-expression relation; and a specific pattern of mtDNA methylation associated with oocyte status/quality that may reflect programming of embryonic development potential.It has been discovered recently that like nuclear genomic DNA (gDNA), mtDNA can be methylated on cytosine residues25. Here, we have optimized a method that includes a linearization step, enabling efficient mitochondrial WGBS26. Because of the size of mitochondrial DNA, we obtained significantly higher coverage of each cytosine (719 nucleoside sites) above 3,000-fold in oocytes samples compared to what is typically obtained for somatic cells . This level of coverage provides extraordinary precision for mitochondrial DNA site-specific analysis and allows comparisons that could lead to better understanding of the precise role of DNA methylation in mitochondria. The purification approach used with granulosa cells was successful in enriching the contribution of mtDNA to the sequencing output by more than 100-fold, albeit with lower coverage to that obtained for oocytes and blastocysts.The existence of mtDNA methylation is still controversial. This can be attributed to the unusual structure of mtDNA coupled with techniques used to study DNA methylationWith the capacity to accurately assess mtDNA methylation, we can now address many important questions about mtDNA methylation in reproductive biology. Thus, for this study, multiple samples were processed to substantiate and compare the effects of environmental exposures and maternal maturity on mtDNA methylation in oocytes, blastocysts and granulosa cells. For our experimental design, mitochondrial WGBS libraries from all samples were pooled in the same sequencing experiment using a multiplex linker, allowing statistical comparisons within and across samples.22, cytosine methylation occurred in multiple contexts, CG, CHG and CHH. As shown in the representative D-Loop region, CHH methylation is the most common compared to CG or CHG in all three sample types  and rns Fig. .Figure 427, we assessed the effects of maternal maturity on mtDNA methylation. Granulosa cells were obtained from hormone-stimulated, large (5–12 mm) follicles of pre-pubertal cows and adult cows. Total cytosine methylation was significantly lower in pre-pubertal heifers compared to adult cows  performed on oocytes and blastocysts for 12 mitochondrial-encoded genes using data from comparable stages to our experiment29.This study is the first to compare mitochondrial DNA methylation in reproductive tissues. We found a more conserved mtDNA methylation signature between oocytes and blastocysts compared to the granulosa cells. This suggests that the oocyte methylation pattern may be inherited by preimplantation embryos. This seems reasonable since mitochondria in embryos are directly inherited from oocytes, with no substantial paternal contribution. Alternatively, CG methylation may be inherited from oocytes and act as nucleation centers for subsequent CHG and CHH methylation in preimplantation embryos. Furthermore, our data indicate different regulatory mechanism between germ/embryonic and somatic cell mtDNA methylation. Consistent with this, comparison of our methylation data with tissues in humans indicates that the observed mtDNA methylation signature associated with granulosa cells is more typical of the somatic environment (% methylation very high or very low at most sites) in comparison to the different patterns observed in oocytes and embryos30. The de novo DNA methyltransferase proteins, DNMT3A and DNMT3L, are likely responsible for non-CG methylation in oocytes. While it is difficult to imagine such proteins entering the mitochondria, it seems equally unlikely that only mitochondrial DNMT1 is responsible for non-CG methylation. Other gene products could be involved in non-replicative  distribution of methylation outside of CG. Of the three currently known DNA methyltransferases, only DNMT1 has been shown to target mitochondria. A recent paper describes a specific isoform of DNMT1 that might enter mitochondria31. Our analysis of RNA bovine sequences present on NCBI indicates a positive mitochondrial localisation sequence within some of the isoforms listed using IPSORT prediction algorithm (http://ipsort.hgc.jp/index.html). Observations in both this study and elsewhere (with mice) suggest that non-CG methylation occurs inside mitochondria and that oocytes would support a less specific catalytic action of such an isoform. In a recent study of melatonin-treated swine oocytes, DNMT1 expression was increased and the protein was trans-located into mitochondria and the inhibitory effect of melatonin on mtDNA expression was suppressed by simultaneous addition of a DNMT1 inhibitor, suggesting that melatonin regulates mtDNA transcription through up-regulation of DNMT1 and mtDNA methylation32. DNMT1 expression is also up-regulated by nuclear respiratory factor 1 (NRF1) and peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC1α), transcription factors that activate expression of nuclear-encoded mitochondrial genes in response to hypoxia, and by loss of tumour suppressor protein 53 (TP53), known to regulate mitochondrial metabolism33. Overall, our investigation suggests a need to increase our understanding of the DNA methyltransferases involved in mitochondria CG as well CGH and CHH methylation in oocytes and preimplantation embryos.In addition to nuclear CG methylation observed in mammals, CHG and CHH methylation account for nearly two thirds of the methyl-cytosines present in mouse germinal vesicles and that these accumulate genome-wide in close proximity to methylated CG sites in growing oocytes34. This shutting down of mitochondria may occurs progressively and could be associated with the general de-methylation of the mtDNA, which could explain why the abattoir oocytes  had higher methylation levels than OS/OPU oocytes (large follicles). Generally, blastocysts possessed a higher level of mtDNA methylation with a broader distribution than oocytes, suggesting subsequent mtDNA methylation programming during embryonic development. However, these levels were reduced in blastocyst produced from abattoir oocytes than OS/OPU oocytes, suggesting that the developmental programming of mtDNA methylation was compromised in the less matured abattoir samples.In this study, we also compared the effects of different ovarian environmental exposures and maternal maturity on mtDNA methylation in oocytes, blastocysts and granulosa cells. We found significant changes in mtDNA methylation in oocytes and blastocysts based on the ovarian environment, which may indicate sensitivity to oocyte status/quality and subsequent mtDNA methylation programming of embryonic development. It is believed that the oocyte metabolism changes during oocyte development as the oocyte mitochondria have a different, immature structure, and are rather quiet compared to somatic tissues35 suggest that they could play a similar role as in the oocyte nuclear genome. Secondly, studies need to be done to analysis mtDNA methylation programming during oogenesis similar to those done for nuclear DNA. This will determine whether the gamete-specific pattern is established initially in early germ cells or is acquired as mitochondria adopt the oocyte-specific form during oocyte growth and maturation. Analysis during preimplantation development will also determine whether mtDNA methylation is inherited from the oocyte to the zygote, and subsequently expanded during preimplantation development. Analyses performed post-implantation will reveal when the demethylated somatic mtDNA methylation pattern, as observed in granulosa cells, is established, as well as whether early lineages or stem cells maintain the embryonic pattern or harbor a somatic program.Many questions remain regarding mtDNA DNA methylation in oocytes and preimplantation embryos. Firstly, in addition of DNA methyltransferases and mtDNA methylation, a greater understanding is required for mechanisms of mtDNA demethylation during gametogenesis. The identification of TET proteins in mitochondria36. Further investigation of various culture environments and medium supplementation with metabolites such as folate will provide further insight into the regulation of mtDNA methylation.Many questions also arise regarding mtDNA DNA methylation and adverse environmental exposures. We observed that suboptimal ovarian environment and maternal maturity were associated with altered mtDNA methylation. Previous studies have revealed that the culture environment of preimplantation embryos not only induced up-regulation of mitochondrial RNA polymerase  at the blastocyst stage, but also increased mitochondrial ND4 mRNA preceding the blastocyst stage, suggesting that impaired regulation of mtDNA may be responsible for the abnormal metabolism and physiology and hence decreased viability of cultured embryos37. Our analysis revealed a difference in mtDNA methylation between the granulosa cells of adult and pre-pubertal cows. These patterns could reflect the linear process of age, or a follicle-specific pattern associated with the ovarian context or the metabolism of a maturing animal compared to an adult. To further explore this observation, follicles of different sizes within the same female should be compared for mtDNA methylation. In cows, the number of mitochondria doubles with age and also during oocyte maturation in vitro38 and exceeding a certain minimal number appears to be necessary for normal early development39. Furthermore, mitochondria methylation has been shown to increase with age in human and in several tissues that have been measured20. To assess linear aging, other tissues within prepubertal and adult females should be compared to assess if the ovary/follicle has different mtDNA methylation programming. Additional studies should examine mtDNA methylation in females of advanced maternal age.Granulosa cells have been studied as a biomarker for oocyte developmental competencede novo promoter methylation, which may have functional consequences on mitochondrial activity and dynamics during early development21. In this study, we examined mtDNA methylation levels and expression of mitochondrial genes. We observed that mtDNA methylation was negatively correlated with individual gene expression. More specifically, genes with higher methylation were associated with lower expression, while genes with low methylation levels had higher expression levels. We also found that gene expression levels were higher in blastocysts compared to oocytes. Higher transcript abundance in blastocysts could be explained by the greater mtDNA copy number in blastocysts (3 times), however, this does not explain the 20 times rise in transcript abundance. Instead, it is likely that the increase of transcription capacity comes from the nucleus in the form of polymerase and transcription factors. Three important transcription regulators, TFAM, Tfb2m and Polrmt, are transcriptional upregulated relative to other genes at the 8-cell stage in bovine embryos , perhaps additionally accounting for the robust expression in blastocysts. Further investigation of somatic tissues, such as liver, muscle and skin that have different mitochondria levels, could be used to study the role of mtDNA during transcription and replication, nuclear-mitochondrial co-regulation, as well as effects of mitochondrial DNA mutations.An important question still revolves around the the potential function of the mtDNA methylation. A recent paper indicated that mitochondrial nuclear genes undergo 40. In pigs, a low-protein diet during pregnancy was associated with sex-dependent epigenetic alterations in the expression of mtDNA-encoded oxidative phosphorylation genes and that the glucocorticoid receptor (NR3C1) is involved in mtDNA transcription regulation41. In humans, mtDNA methylation is associated with early insulin sensitivity and body mass index. An increase in BMI from lean to obese (20–24.9 to 30–34.9) coincides with a drastic increase in mtDNA methylation, while increases from obese to severely obese (35–39.9) and from severely to morbidly obese (BMI > 40) coincide with marginal increases in methylation42. Further investigations are required to determine the linkages between mtDNA methylation, mitochondrial function and cellular metabolism.In a broader context, the major role of mitochondria is to provide ATP, and their functionality is therefore closely related to metabolism. With respect to reproductive biology, mtDNA sequences encoding RNR1, RNR2 and ND4 as well as the D-loop region have been found significantly hypermethylated in porcine oocytes in association with PCOS, indicating that abnormal activation of one-carbon metabolism and hypermethylation of mtDNA may contribute substantially to mitochondrial malfunction and decreased oocyte qualityThe results presented here show that mtDNA methylation patterns in bovine oocytes and embryos differ from those in the associated granulosa cells and therefore warrant investigation of how such patterns control replication and transcription of mtDNA in both embryonic and somatic tissues. Secondly, the observed differences in oocyte quality and reflected in blastocyst methylation patterns may be indicative of late oogenesis/folliculogenesis programming, which likely has an impact on embryo quality or on the metabolic programming of the foetus or of the offspring.43 (5–12 mm follicles) in peri-pubertal animals (8–16 months old) obtained from a commercial IVF company . The procedure allows for oocyte to remain in the growing follicle under FSH (Follicle stimulating hormone) stimulation which become dominant after a few days increasing the quality of the enclosed oocytes compared to oocyte collected from random non-stimulated animal at slaughter. Both types of oocytes are at the same maturation stage (GV) at collection but if exposed to similar culture conditions will lead to blastocysts rates of >50% for the stimulated compared to <30% for the abattoir derived44. Day-7 blastocysts were in vitro produced using in vitro maturation, fertilization and embryo development from abattoir GVO45 or OPU GVO37. Granulosa cells were obtained from hormone-stimulated 113-month-old  or 6-month-old  cows by trans-vaginal ultrasound aspiration44. Experiments took place in compliance with the guidelines of the Canadian Council on Animal Care and supervised by the Animal Protection Committee of Université Laval. These guidelines are strictly followed by Boviteq, who provided all the tissues and samples. The study did not require handling of animals on university premises.Pools (n = 10) of germinal vesicle oocytes (GVO) were collected from 2–5 mm follicles from ovaries obtained from randomly selected animals at a local abattoir and also from females subjected to ovarian stimulation protocol (OS) followed by trans-vaginal ultrasound aspiration through a process called ovum pick-up (OPU)25. Briefly, cells were suspended in 500 µL of RSB  hypotonic buffer in a 1.0 mL glass homogenizer tube using 10 strokes in a B-pestle, diluted with 350 µL of 2.5X MS buffer, and then centrifuged at 1,300 × g for 5 min at 4 °C. The pellet was resuspended, washed with 500 µL of 1X MS buffer and spun again , following which the two supernatants were pooled in a clean tube and centrifuged at 16,000 × g for 15 min at 4 °C. The pellet was re-suspended in 500 µL of 1X MS buffer and washed . Mitochondrial DNA was extracted from the final pellet using ZR Genomic DNA – Tissue MicroPrep (Zymo Research) and then suspended in 40 µL of DNA elution buffer. MtDNA was digested with plasmid-safe ATP-dependent DNase (Epicenter) in a final volume of 50 µL for 2 h at 37 °C followed by 30 min at 70 °C, and then purified using ZR Genomic DNA – Tissue MicroPrep without proteinase K digestion. The purified DNA was suspended in 10 µL of DNA elution buffer.The vast mitochondrial abundancy in oocytes and preimplantation embryos  does not require mitochondrial enrichment prior to mtDNA purification. For granulosa cells, an enrichment procedure was use where mitochondria were isolated with hypotonic buffer followed by differential centrifugation26, a restriction enzyme was used  to linearize the bovine mtDNA samples. Next, samples were treatment with 130 µL of Lightning Conversion Reagent from the Pico Methyl-Seq Library Prep kit (Zymo Research). The WGBS library protocol was followed as recommended by the manufacturer for 10 pg to 1 ng of DNA with 10 cycles for the final two PCR amplification rounds. Purified libraries were suspended in 12 µL of DNA elution buffer.Pools of 10 GVO or 10 blastocysts were digested with proteinase K for 20 minutes at 50 °C in M-digestion buffer (1X) to obtain a final volume of 20 µL. Granulosa cell mtDNA was mixed with 10 µL of 2X M-digestion buffer and brought to a final volume of 20 µL using the reagents from the EZ DNA Methylation-Direct kit (Zymo Research). Since it has been reported that accessibility of the human mtDNA nucleoid to sodium bisulphite varies can compromise the mtDNA methylation analysisSix libraries prepared with the Pico Methyl-Seq Library Prep kit (Zymo Research) and the Illumina Index linker-primers for multiplexing (Illumina) were quantified, pooled and sequenced on one lane of Hiseq2500 SR 50 bp (Illumina). Library quantitation, quality control and sequencing protocol were performed at the McGill University and Génome Québec Innovation Centre . The sequences were analysed against the reference bovine mitochondrial bisulphite-treated DNA, and the ratio of methylation was analysed for each individual site, and for the 3 types of cytosine positions . A Fisher-type analysis was performed with false discovery rate (FDR) filtering. The data discussed in this paper have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) and are accessible through GEO Series Accession number GSE122599.46. Reads with a minimal length of 50 nucleotides were kept for further processing. Methylation calls for the bovine mitochondrial genome (NC_006853.1) were then obtained using bismark47.Using Trimmomatic, sequencing adapters were removed and base calls with a quality score below 30 were removed from the ends of the readsin vitro as described previously were treated with sodium bisulphite directly using the EZ DNA Methylation-Direct kit. PCR was performed for 2 regions on mtDNA, namely the D-loop and ND4L, in 25 µL using the PyroMark PCR kit (Qiagen) in 1X buffer, 3 mM MgCl2 and 5 µL bisulphite-modified DNA (corresponding to 0.1 blastocyst). Primers information are presented in Suppl Table To validate WGBS cytosine methylation results, we performed pyro-sequencing for several methylation sites in the D-Loop and ND4L regions of the mitochondrial genome. Individual blastocysts (n = 6) produced in vitro from these GVOs were extracted using ZR Genomic DNA – Tissue MicroPrep for gDNA and mtDNA. Primers for the D-loop region on mtDNA (Suppl Table http://www.idtdna.com/Scitools/Applications/Primerquest/) from sequences obtained using the Genebank V00654.1 Bos taurus complete mitochondrial DNA. To confirm primer specificity, the amplified fragment was purified by electrophoresis on a standard 1.2% agarose gel then quantified and sequenced. The products were then used to create the standard curve for quantification, with dilutions ranging from 2 × 10−4 to 2 × 10−8 ng/µL. Realtime PCR was performed on a LightCycler 480 (Roche Diagnostics) using LightCycler 480 SYBR Green I Master. Each qPCR reaction mixture  contained the mtDNA from single GVOs or blastocysts. The PCR conditions were as follows: denaturing cycle for 10 min at 95 °C; 50 PCR cycles ; and a final cooling step at 40 °C. Quantification was performed with LightCycler 480 Software version 1.5 (Roche Diagnostics) by comparison to the standard curve. PCR specificity was confirmed by melting-curve analysis. Mitochondrial DNA copy numbers were calculated from the standard curve.Pools of 20 GVO (n = 3) collected from abattoir and 7-day blastocysts (n = 3) produced in vitro28. Briefly, the libraries were prepared with Ovation RNAseq v2 kit (NuGEN) and the barcoded libraries were pooled for multiplexed sequencing on an Illumina GAIIx to a mean coverage of 20 × 106 reads each. Sequencing runs were done in single-read mode with an 80-base read length.For the level of expression of mitochondrial genes, 12 mitochondrial transcripts abundance were obtained from GEO Series GSE52415 acquired from sequencing pools of 10 denuded bovine oocytes and 10 blastocysts produced All statistical analyses were performed using Graphpad prism 5 software. An unpaired t-test analysis was performed for total mtDNA methylation, mtDNA methylation at specific cytosines, and mtDNA abundance. Anova analysis was performed for the mRNA gene-specific expression analysis and a Pearson r correlation analysis for the methylation/reads analysis.Supplemental material


Drosophila model harboring an incompatibility between a mitochondrial tRNAtyr and its nuclear-encoded mitochondrial tyrosine synthetase to assess the cellular mechanisms affected by this incompatibility and to test the relative contribution of mitonuclear interactions and aging on the expression of impaired phenotypes. Our results show that the mitochondrial tRNA mutation caused a decrease in mitochondrial oxygen consumption in the incompatible nuclear background but no effect with the compatible nuclear background. Mitochondrial DNA copy number increased in the incompatible genotype but that increase failed to rescue mitochondrial functions. The flies harboring mismatch between nuclear and mitochondrial genomes had almost three times the relative mtDNA copy number and fifty percent higher rate of hydrogen peroxide production compared to other genome combinations at 25 days of age. We also found that aging exacerbated the mitochondrial dysfunctions. Our results reveal the tight interactions linking mitonuclear mismatch to mitochondrial dysfunction, mitochondrial DNA regulation, ROS production and aging.Mitochondrial and nuclear genomes have to coevolve to ensure the proper functioning of the different mitochondrial complexes that are assembled from peptides encoded by both genomes. Mismatch between these genomes is believed to be strongly selected against due to the consequent impairments of mitochondrial functions and induction of oxidative stress. Here, we used a  Dysfunctional mitochondria are thought to be a proximal mechanism for aging due to the dual role of mitochondria as a source and target of reactive oxygen species (ROS) associated with aging . In mitoIt has been suggested that long-lived species do not tolerate mismatch between nuclear and mitochondrial genomes because of the importance of fine tuning aerobic metabolism and the role of ROS generation (and buffering) in the induction of apoptosis . The natDrosophila species. Specifically, mtDNAs from either D. melanogaster (ore) or D. simulans (501simw) were substituted into two D. melanogaster wild-type nuclear backgrounds, OreR and Aut (501simw) mitochondrial genome differs from the (ore) at the level of a tRNAtyr single nucleotide polymorphism (SNP), whereas the Aut nuDNA differs from the OreR by a SNP in a mitochondrial tyrosine synthetase. The (501simw);OreR combination is characterized by multiple deleterious effects on development, reproduction, locomotion, respiration of isolated mitochondria and mitochondrial morphology due to an incompatibility between (501simw) mitochondrial tRNAtyr and the OreR nuclear-encoded mitochondrial tyrosine synthetase  from D. melanogaster or (501simw) from D. simulans siII haplotypes, and by replacing the nuclear genomes with either OreR or Aut chromosomes using non-recombining balancer chromosomes, and were the same as previously studied by others (501simw);OreR mitonuclear genotype harboring the incompatibility have a mean lifespan as low as 32 days depending on the diet (D. melanogaster is more pronounced and results in decreased longevity and increased rate of senescence (n = 12) for all the experiments except for the measurement of H2O2 production in isolated mitochondria in which six different pools of 30 flies were used (n = 6). On each experimental day, thoraces were dissected and either directly processed for mitochondrial isolation, high-resolution respirometry, and confocal microscopy or immediately stored at -80°C for DNA extraction and biochemical analysis.Drosophila lines were provided by D. Rand and were constructed by introgressing mitochondrial genomes i.e.,  and reverse (AGAAACCAACCTGGCTTACACCGGTTTGAACTCAG for mtDNA gene and GGCTATGGAGTCGGTGATGG for nuDNA gene) primers, 0.3 U of Taq polymerase and 30 ng of DNA. Each DNA fragment was verified on 1.5% agarose gel. Cleaning and sequencing of PCR products were conducted at McGill University and Génome Québec Innovation Centre . All DNA sequences were then analyzed in Sequencher® version 4.9 sequence analysis software.Genomic DNA was extracted from 3 thoraces using E.Z.N.A.Sequences obtained from the mitochondrial and nuclear genomic DNA of the different genotypes were aligned using MEGA version 5.1 . Each DN® No-ROX mix, 1 ng of DNA, 0.2 μM of each forward (CAACCATTCATTCCAGCCTTC for mtDNA gene and AGGCGTTTGAGTGGTTGG for nuDNA gene) and reverse (GTCTAACCTGCCCACTGAAA for mtDNA gene and CGCTTTGGGCTTTTTGGAT for nuDNA gene) primers. Reactions were run in triplicates on a LightCycler® 480 Real-Time PCR System using the following thermal profile: 95.0°C for 3 s, followed by 40 cycles of 95.0°C for 30 s, 60.0°C for 30 s, and 72°C for 30 s. The efficiency of each primer set (Enu and Emt) was determined using appropriate serial dilutions before sample analyses and primer specificity for each gene was verified by regular PCR and dissociation curve analysis following qPCR protocols. Copy number of the mtDNA gene relative to the nuDNA gene was calculated using the cycle thresholds (Ct) for each gene and kinetic PCR efficiency correction with the following formula: 2∗(EnuCtnu/EmtCtmt).The relative mtDNA copy number per diploid nuclear nuDNA genome was evaluated as previously described . MitochoDrosophila  of the complex(es) involved followed by the state of respiration (complex-STATE) as previously described (Thorax muscles (3 thorax for each measurement) were permeabilized at 4°C using BIOPS relaxing solution  complemeosophila . They weescribed . After mescribed .® Green FM and 5 μM of CellROX® Deep Red Reagent  for 30 min at 24°C in order to label mitochondria and oxidation by ROS, respectively. Labeled fibers were then observed with a Zeiss LSM 700 confocal laser scanning microscope coupled to an Axio Observer inverted microscope . Images were acquired in 8 bits using LD Plan-Neofluar 40x/0.6 Korr M27 objective and captured using ZEN 2012 1.1.1.0 . Excitation/emission wavelength were λexc/λem = 488/BP 420–550 nm and λexc/λem = 639/LP 640 nm for Mitotracker® Green FM and CellROX® Deep Red Reagent, respectively. 20 z-stack confocal images  of fibers were acquired using z-slices of 0.91 – 1.79 μm with pinhole size of 70.14 μm. Image J software  was used to analyze and quantify fluorescence in confocal images. For quantification, z-projections of 10 raw confocal images were generated and were processed using the “Autothreshold Default” method of Image J. Fluorescence measurements in the resulting images were performed using the “Integrated Density” option [which corresponds to the mean fluorescence value in the region of interest (ROI) multiplied by the ROI area] and were corrected with background readings. Results are expressed as arbitrary units of fluorescence intensity.Single muscle fibers from thorax were dissected in BIOPS relaxing solution  and incu® red reagent (Invitrogen) and an EnVision® microplate reader  set at 24°C with excitation/emission set at 560/587 nm. For each sample, maximal H2O2 production at the level of complex I , as well as at the level of complexes I and III  were measured. Results are presented as means ± s.e.m. of nmol H2O2 produced per minute per mg of proteins.Intact mitochondria were isolated at 4°C as previously described  using 30® microplate reader  set at 24°C as previously described  and lipids  using commercially available kits from Cayman Chemicals  and following the manufacturer’s protocol.Activity of NADH:ubiquinone oxidoreductase (complex I) and ubiquinone:cytochrome c oxidoreductase (complex III), CI+CIII, as well as citrate synthase (CS) activity were evaluated in crude homogenates of thorax (pool of 3 flies) using an EnVisionescribed . CI+CIIIescribed  by folloescribed  and are P-values (Tukey method) with significance set at P<0.05. Normality and homogeneity of variance were verified beforehand using Shapiro–Wilk and Levene’s tests, respectively, and data were ln-transformed when required. F-ratios and significance from three-way ANOVAs on all the different parameters studied are presented in All statistical analyses were performed with the R software . For all parameters, three-way ANOVAs considering MtDNA, NuDNA and Age as fixed factors were performed. When an interaction  revealed a significant effect, multiple comparisons were tested with pairwise comparisons of the least-squares means using adjusted 501simw);OreR flies had an almost three-fold increase in mtDNA copy number compared to (ore);Aut, (ore);OreR, and (501simw);Aut when evaluated at both 15 and 25 days old ;OreR compared to the other lines at 15 days old ;OreR and (ore);Aut. To validate these results obtained with confocal microscopy, we also evaluated the CS activity as a proxy of mitochondrial content (501simw);OreR compared to the other lines at 15 days old ;OreR ;OreR at both ages, this was not significantly different than the other genotypes (501simw);OreR at 25 days old with 23.19 ± 0.84 μmol.mg-1 proteins, but it was not significantly different from the other genotypes at the same age .Oxidative stress was assessed by the fluorescent marker CellROXe fibers  and was e fibers . Althougenotypes . As an aenotypes . The hig2O2 production derived from the production of superoxide anion in isolated mitochondria. Both conditions tested, i.e., maximal H2O2 production at the level of complex I and at the level of complexes I + III, displayed the same pattern i.e., effects of MtDNA, NuDNA, Age as well as of the interactions MtDNA × NuDNA and MtDNA × Age ;Aut and (501simw);OreR ;OreR, H2O2 production was significantly higher compared to the other mitonuclear genotypes ;Aut compared to the genotypes harboring the (ore) mtDNA (P < 0.001 and P = 0.02 for (ore);Aut and (ore);OreR, respectively) and (501simw);OreR also presented lower CI-LEAK than (501simw);Aut ;OreR presented significantly lower respiration rates than (ore);Aut for CI-OXPHOS (P = 0.02) and Complex IV (P = 0.03) when measured at 15 days old ;Aut and (501simw);OreR presented significantly lower CI-OXPHOS than (ore);Aut and (ore);OreR ;OreR was still lower but not (501simw);Aut ;OreR than in the other lines at both ages ;Aut and (501simw);OreR] causes a decrease in oxygen consumption when mitochondrial respiration is supported by electron entrance at the level of complex I; (ii) at the same age, this decrease is alleviated upstream of complex III by an increase in respiration rate owing to the nuclear encoded mG3PDH in the mitonuclear combination (501simw);Aut; (iii) in the (501simw);OreR which harbors the incompatibility, a strong increase in mitochondrial DNA copy number was observed (a process likely triggered by an observed increase in H2O2 production), leading to mtDNA excess which could indicate replication errors. We also found that aging has more effects on the genotypes with the (501simw) mtDNA than those with the (ore) as shown by an increase in maximal H2O2 production by complex I and complexes I and III, and a decrease of CI-OXPHOS states. Specifically, the (501simw);OreR genotype is affected at an early age, and Drosophila with the (501simw);Aut genotype are more affected in terms of H2O2 production at the level of complex I when measured at 25 days old compared to 15 days old.In this study, we have thoroughly analyzed mitochondrial phenotypes harboring different mitonuclear combinations in flies at two different ages. Our results show that: (i) at 25 days old, a mitochondrial tRNA mutation expressed in two different nuclear backgrounds  or an incompatible  associated amino-acyl-tRNA synthetase. The decreased CI+CIII enzymatic activity and respiration rates observed in (501simw);OreR flies at both ages but not in the other combinations tested seems to be a major consequence of the incompatibility between (501simw) mitochondrial tRNAtyr and OreR tyrosine-tRNA synthetase. This result is consistent with other studies showing decreased enzymatic activities of individual ETS complexes encoded by both genomes  in (501simw);OreR and suggests an impairment of mitochondrial functions that leads to major consequences at the phenotypic level ;Aut presented reduced mitochondrial oxygen consumption when assayed with complex I substrates (CI-LEAK and CI-OXPHOS) to the same extent as (501simw);OreR. It suggests that the mitochondrial tRNAtyr SNP and therefore the mitochondrial genome has an impact on oxygen consumption by mitochondria via the input of electrons through the ETS, but that this effect is more important in 25 days old flies. Considering that complex I has the highest number of mitochondrial subunits of all ETS complexes (7 subunits), our results suggest major consequences of any impairment in mtDNA translation and transduction of mitonuclear exchanges on functional properties of complex I. Morevover, the depressed CI mitochondrial functions found in 25 days old (501simw);Aut suggest a high sensitivity to mutation accumulation with aging in the mitochondrial genes encoding this complex. This is clearly illustrated by the strong impact of the MtDNA and Age factors in the statistical model at the level of mitochondrial respiration when electrons are exclusively supplied to complex I. Of relevance, complex I also contains the most tyrosines (98) which implicates a major effect of the tRNA-synthetase interaction on the mitochondrial functions ;Aut, with respiration rates reaching the same level as (ore);Aut and (ore);OreR of the same age. Although the mitochondrial tRNA SNP is linked to OXPHOS deficiencies, (501simw);Aut flies are able to bypass these deficiencies when higher ETS capacity is mobilized upstream of complex III via mG3PDH activity. The mG3PDH is at the crossroads of glycolysis, oxidative phosphorylation, and fatty acid metabolism, and is fundamental for NADH homeostasis in the cytosol (Increasing the electron flux in the ETS by supplying G3P (CI+mG3PDH-OXPHOS) resulted in a complete compensation in 25 days old ( cytosol . Therefo501simw);OreR at both ages, suggesting a strong response at the level of mtDNA regulation. This could reflect an increase of mitochondrial biogenesis, and thus of mitochondrial content. Indeed, increased mtDNA copy number is usually associated with mitochondrial biogenesis (501simw);OreR, we did not detect evidence for increased mitochondrial biogenesis, as mitochondrial content remains significantly lower than in the other genotypes , highly contrasting with the results on relative mtDNA copy number. The strong response in mtDNA seems to be a consequence of the mitochondrial dysfunctions that possibly participate in mutation accumulation, leading to the observed phenotype ;OreR are producing more ROS than the other genotypes and that the genotypes harboring the (501simw) mitochondrial DNA have increased ROS production with aging. However, this increased ROS production did not translate into oxidative damages to proteins or lipids. An explanation for these results would be that the carbonyl and TBars assays used in our study are not sensitive enough to detect oxidative damages to proteins and lipids, respectively, and that other markers such as thiol and 4-hydroxynonenal assays might be worth to measure as an alternative. However, (501simw);OreR flies displayed slightly higher TBars content when both ages were combined , suggesting slightly higher oxidative stress in this genotype.We found an almost three fold increase in the mtDNA copy number in ;OreR despite increased mtDNA copy number, which is usually associated with mitochondrial biogenesis (501simw);OreR is due to mitophagy rather than to an impairment of biogenesis. Indeed, our results show similar pattern  to what has been observed in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) fibroblasts, which is a disorder caused by mutations in mitochondrial tRNA genes (501simw);OreR as well as structural mitochondrial abnormalities (loose cristae structure and matrix gaps) compared to the other genotypes detected by It is surprising that mitochondrial content was lower in (ogenesis . Simultaogenesis . These togenesis . It is tNA genes . This is501simw);Aut, the mG3PDH which is a nuclear-encoded complex of the ETS can participate in the maintenance of mitochondrial oxidative capacities by compensating for a complex I deficiency. When a mutation in the corresponding nuclear amino-acyl-tRNA synthetase occurs, as in the (501simw);OreR genotype, a mitonuclear mismatch appears. This results in a reduced catalytic capacity at the level of CI+CIII and diminished respiration rates likely leading to a more reduced state of ETS upstream of complex IV and inflation of maximal H2O2 production. This response can promote the strong increase observed in mtDNA copy number. These two concomitant responses (higher ROS production and mtDNA excess) could increase mutation accumulation that, in turns, exacerbate mitochondrial dysfunctions. Introgressed hybrids can however survive mitonuclear mismatch, even if growth and lifespan are clearly compromised ;OreR genotype ;OreR had lower survival under infection with a natural pathogen, with females experiencing immunity–fecundity tradeoffs. While the impact of defective ETS on ROS mitochondrial efflux was clear in our study, we have not been able to detect any associated trace of oxidative stress. This suggests that either the mechanisms involved in buffering ROS were adequate to prevent any damage or that our markers did not provide the necessary resolution to detect significant ROS injuries. In the TBars assay for example, thiobarbituric acid does not only react with the products of lipids peroxidation such as malondialdehyde and is at best a weak marker of ROS attack on lipids. Further studies using more sensitive techniques, such GC-MS, are needed to precisely monitor some specific products of lipid peroxidation such as 4-Hydroxynonenal or 4-Hydroxyhexenal.The role of ROS in the aging process is still debated, mostly in short-lived invertebrates such as drosophila and nematodes, but our results confirm that the reduction in average lifespan observed in the  over a wider range of lifespan to detect key characters susceptible to set physiological and longevity limitation.Another limitation of our study is that we characterized the cellular response following mitonuclear mismatch in flies at only two different biological ages. The two chosen ages (15 and 25 days old) are however not usually considered as advanced in NP and PB designed the research. NP, GC, and RB performed the research. FD and PB provided the materials for experiments. DR provided the different genotypes. NP analyzed the data and wrote the first draft of the manuscript. All authors participated in the subsequent versions of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor and reviewer NJ declared their involvement as co-editors in the Research Topic, and confirm the absence of any other collaboration.


In both a human epidermal cell line, HaCaT, and a mouse melanoma B16F10 cell line, we found that tRA could activate SerRS transcription through binding with the SerRS promoter. However, UV irradiation induced activation of ATM-phosphorylated SerRS, leading to the inactivation of SerRS as a transcriptional repressor of vascular endothelial growth factor A (VEGFA), which dampened the effect of tRA. When combined with ATM inhibitor KU-55933, tRA showed a greatly enhanced efficiency in inhibiting VEGFA expression and a much better protection of mouse skin from photo damage. Also, we found the combination greatly inhibited tumor angiogenesis and growth in mouse melanoma xenograft in vivo. Taken together, tRA combined with an ATM inhibitor can greatly enhance the anti-angiogenic activity of SerRS under UV irradiation and could be a better strategy in protecting skin from angiogenesis-associated skin damage and melanoma caused by UV radiation.Retinoic acid (RA) has been widely used to protect skin from photo damage and skin carcinomas caused by solar ultraviolet (UV) irradiation, yet the mechanism remains elusive. Here, we report that all-trans retinoic acid ( Excessive exposure to sunlight causes sunburn, primarily because the short wavelength 280–320 nm) ultraviolet (UV)-B irradiation causes skin damage . Acute s80–320 nmtRA) is the first generation of retinoids. It has been adopted in clinical use for several decades and still is popular for its low cost. Retinoic effects on photo-damaged skin include improvement of coarse wrinkling and dyschromia, as well as decreased roughness and vascularization. However, the efficiency of monotherapy is limited; furthermore, the toxicity and teratogenicity would arise along with the increase of concentration and extension of treatment period  vector (between Kpn I and Xho I sites) to create the pGL4–SerRS–WT, pGL4–SerRS–△RARE1, and pGL4–SerRS–△RARE2 firefly luciferase reporter plasmids. After 16 h of incubation in 12-well plates, HaCaT cells were transiently transfected with 100 ng of the aforementioned reporter plasmids, and 500 ng of the pFlag–CMV-2 empty vector was transfected as the control. A Renilla luciferase control reporter plasmid pRL-SV40 (50 ng) was co-transfected to normalize the transfection efficiency among different experiments. Six hours after transfection, the cells were treated with DMSO or tRA in inhibiting UV-induced VEGFA expression and angiogenesis. Taken together with this study, our findings contribute to a new understanding of UV irradiation on human skin and provide a promising approach for the prevention of angiogenesis-related skin damage and melanoma progression by targeting ATM and SerRS.In summary, this study has revealed a novel signaling cascade that controls VEGFA expression upon UV exposure and is mediated by the ATM–SerRS pathway. Moreover, we discovered the undetected mechanism of


Plasmodium berghei, we show that the alveolin domain is responsible for targeting IMC1h to the membrane skeleton and, consequently, its deletion from the protein results in loss of function manifested by abnormally-shaped ookinetes and sporozoites with reduced tensile strength, motility and infectivity. Conversely, IMC1h lacking its non-alveolin conserved domain is correctly targeted and can facilitate tensile strength but not motility. Our findings support the concept that the alveolin module contains the properties for filament formation, and show for the first time that tensile strength makes an important contribution to zoite infectivity. The data furthermore provide new insight into the underlying molecular mechanisms of motility, indicating that tensile strength is mechanistically uncoupled from locomotion, and pointing to a role of the non-alveolin domain in the motility-enhancing properties of IMC1h possibly by engaging with the locomotion apparatus.Invasive, motile life cycle stages (zoites) of apicomplexan parasites possess a cortical membrane skeleton composed of intermediate filaments with roles in zoite morphogenesis, tensile strength and motility. Its building blocks include a family of proteins called alveolins that are characterized by conserved “alveolin” domains composed of tandem repeat sequences. A subset of alveolins possess additional conserved domains that are structurally unrelated and the roles of which remain unclear. In this structure-function analysis we investigated the functional contributions of the “alveolin” vs. “non-alveolin” domains of IMC1h, a protein expressed in the ookinete and sporozoite life cycle stages of malaria parasites and essential for parasite transmission. Using allelic replacement in  Plasmodium species, the causative agents of malaria, have a complex life cycle in vertebrate host and mosquito vector. Parasite-infected erythrocytes multiply via an asexual replication cycle called schizogony to release merozoites that infect new red blood cells. A small percentage of these develop into sexual stage precursor cells (gametocytes) which, after uptake with the blood meal of a feeding mosquito, begin a rapid process of gamete formation and fertilization inside the mosquito midgut. The resultant zygotes undergo meiosis and transform into elongated forms termed ookinetes, which traverse the midgut epithelium and then round up to form the oocysts. In the following weeks, young oocysts grow and divide by a process known as sporogony to generate hundreds of daughter cells named sporozoites. After egress from the oocyst, sporozoites invade and inhabit the salivary glands, and are transmitted to new hosts by mosquito bite to first infect liver cells from which new malaria blood stage infections are initiated to complete the life cycle.Plasmodium life cycle. These so-called “zoite” stages possess a characteristic cortical structure termed the pellicle. The pellicle is defined by a double membrane structure termed the inner membrane complex (IMC) situated directly underneath the plasma membrane, which is equivalent to a sutured system of flattened sacs or alveoli  primers IMC1hdeltadomain1-F (GAGGTTCACAATATTTGAATAACAATCAAGCACA) and IMC1hdeltadomain1-R (AAATATTGTGAACCTCCATACAAAGTGTGTT) were used to PCR amplify plasmid pLP-IMC1h/GFP  and P3 (GTGCCCATTAACCATCACC)   . The absAAATGTG) .t-test.Images of individual midgut sporozoites were captured by microscopy on Zeiss LSM510 inverted laser scanning confocal microscope. Using Zeiss LSM image browser software the circumference was measured from which the occupied surface area (“footprint”) was calculated as a measure of cell size. Statistical analysis was carried out using Student's Ookinete motility was assessed essentially as previously described  were removed if desired. For sporozoite transmission, the prevalence of infection of mosquito batches was determined and combined with the number of blood meal-positive insects after feeding to estimate the number of sporozoite-infected mosquitoes that fed.At 6 days before infecting mosquitoes, mice were injected intraperitoneally with phenylhydrazine  to induce reticulocytosis. At 3 days before mosquito feeding, mice were infected intraperitoneally with 10Plasmodium species clearly reveals the presence of its single conserved “alveolin” module (domain 1), as well as a conserved carboxy-terminal module that is structurally unrelated (domain 2)   reduced oocyst numbers compared to IMC1h/GFP control parasites  of IMC1hΔ2 midgut sporozoites had a normal crescent shape and displayed cortical localization of GFP fluorescence (P < 0.001), which in turn were significantly smaller than IMC1h/GFP sporozoites (P < 0.005) . Interestingly, IMC1hΔ2 sporozoites lost their normal shape during transition from the midgut to the salivary glands, resulting in salivary gland sporozoites possessing a bulging area , and had similar efficacy to IMC1h/GFP sporozoites in colonizing the salivary glands  and more meandering movement patterns  Act 1986 implementing European Directive 2010/63 for the protection of animals used for experimental purposes.JD and CV contributed conception and design of the study. MC, AT, SS, and JD performed experiments and interpreted results. MC wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Loss of arm function is a common problem after stroke. Robot-assisted training might improve arm function and activities of daily living. We compared the clinical effectiveness of robot-assisted training using the MIT-Manus robotic gym with an enhanced upper limb therapy (EULT) programme based on repetitive functional task practice and with usual care.RATULS was a pragmatic, multicentre, randomised controlled trial done at four UK centres. Stroke patients aged at least 18 years with moderate or severe upper limb functional limitation, between 1 week and 5 years after their first stroke, were randomly assigned (1:1:1) to receive robot-assisted training, EULT, or usual care. Robot-assisted training and EULT were provided for 45 min, three times per week for 12 weeks. Randomisation was internet-based using permuted block sequences. Treatment allocation was masked from outcome assessors but not from participants or therapists. The primary outcome was upper limb function success (defined using the Action Research Arm Test [ARAT]) at 3 months. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN69371850.Between April 14, 2014, and April 30, 2018, 770 participants were enrolled and randomly assigned to either robot-assisted training (n=257), EULT (n=259), or usual care (n=254). The primary outcome of ARAT success was achieved by 103 (44%) of 232 patients in the robot-assisted training group, 118 (50%) of 234 in the EULT group, and 85 (42%) of 203 in the usual care group. Compared with usual care, robot-assisted training (adjusted odds ratio [aOR] 1·17 [98·3% CI 0·70–1·96]) and EULT (aOR 1·51 [0·90–2·51]) did not improve upper limb function; the effects of robot-assisted training did not differ from EULT (aOR 0·78 [0·48–1·27]). More participants in the robot-assisted training group (39 [15%] of 257) and EULT group (33 [13%] of 259) had serious adverse events than in the usual care group (20 [8%] of 254), but none were attributable to the intervention.Robot-assisted training and EULT did not improve upper limb function after stroke compared with usual care for patients with moderate or severe upper limb functional limitation. These results do not support the use of robot-assisted training as provided in this trial in routine clinical practice.National Institute for Health Research Health Technology Assessment Programme. Upper limb problems commonly occur after a stroke, comprising loss of movement, coordination, sensation, and dexterity, which lead to difficulties with activities of daily living (ADL) such as washing and dressing. About 80% of people with acute stroke have upper limb motor impairment, and of those with reduced arm function early after stroke, 50% still have problems after 4 years.How to optimise stroke patients' upper limb recovery is unclear. Systematic reviews of therapy interventions suggest that patients benefit from therapy programmes in which they practise tasks directly rather than from interventions that focus on impairments.Robot-assisted arm training has shown promise for improving ADL, arm function, and arm muscle strength after stroke.Evidence before this studyRobot-assisted training enables stroke patients with moderate or severe upper limb impairment to perform repetitive tasks in a highly consistent manner, tailored to their motor abilities. A 2018 Cochrane review  reported that electromechanical and robot-assisted arm training improved arm function (standardised mean difference [SMD] 0·32 [95% CI 0·18–0·46]) and activities of daily living (ADL) scores (0·31 [0·09–0·50]). The clinical importance of these differences is unclear and the results should be interpreted with caution because of study heterogeneity and variation in the quality of the included studies. Another review found no difference in any of these outcomes when robot-assisted training was compared with the same duration or intensity of conventional therapy. Theories of neuroplasticity and motor learning support an approach to rehabilitation based on repetitive practice of functional tasks. A 2016 Cochrane review  found that repetitive functional task practice for patients who had a stroke was associated with improved arm function (SMD 0·25 [95% CI 0·01–0·49]), hand function (0·25 [0·00–0·51]), and ADL (0·28 [0·10–0·45]). Again, the clinical importance of these findings is unclear.Added value of this studyRATULS is the first multicentre trial with adequate statistical power to compare robot-assisted training with both an evidence-based therapy programme of the same frequency and duration  and usual care. We found no significant difference in our primary outcome of upper limb function at 3 months between stroke patients treated with robot-assisted training, an enhanced upper limb therapy programme (EULT), or usual care. Although some improvements for robot-assisted training were observed in upper limb impairment compared with usual care, these did not translate into improvement in upper limb function or ADL. EULT resulted in less upper limb impairment, better mobility, and better ADL at 3 months compared with usual care. Furthermore, EULT was superior to robot-assisted training for ADL at 3 months.Implications of all the available evidenceThe results of the RATULS trial do not support the routine use of robot-assisted training (MIT-Manus robotic gym) for patients with moderate or severe upper limb functional limitation resulting from stroke. Our findings will inform guidelines about the use of robot-assisted training and EULT in clinical practice. Further research is needed to find ways to translate the improvements in upper limb impairment seen with robot-assisted training into improvements in upper limb function and ADL. This might involve combining robot-assisted training with more functionally orientated therapy strategies. The RATULS trial provides evidence of the potential benefit of EULT, although as delivered in this trial, it is unlikely to be cost-effective. Innovations to make enhanced rehabilitation programmes more clinically effective and cost-effective are needed.This randomised controlled trial (RCT) aimed to establish whether robot-assisted training improved upper limb function after a stroke compared with an enhanced upper limb therapy (EULT) programme of the same frequency and duration and usual care alone. Robot-assisted training and EULT involved repetitive task practice and were provided in addition to usual care.RATULS was a three-group, pragmatic, multicentre RCT done at four National Health Service (NHS) centres in the UK. Each centre comprised a stroke service in an NHS hospital with an MIT-Manus robotic gym system , plus stroke services in adjacent NHS Trusts and community services. The trial protocol was approved by National Research Ethics Committee Sunderland (reference 13/NE/0274) and has been published.Study participants were adults (age ≥18 years) with moderate or severe upper limb functional limitation (Action Research Arm Test [ARAT] score 0–39)Randomisation was done through a central independent web-based service hosted by Newcastle University Clinical Trials Unit. Participants were randomly assigned 1:1:1 to receive robot-assisted training, an EULT programme, or usual care using permuted block sequences stratified according to centre, time since stroke, and severity of upper limb functional limitation (ARAT score).Robot-assisted training and EULT programmes were delivered at the same frequency and duration: 45 min of face-to-face therapy, three times per week for 12 weeks. The same therapists and therapy assistants delivered both interventions at each centre. A detailed description of robot-assisted training and EULT using the Template for Intervention Description and Replication checklist is available.The robot-assisted training programme integrated training with all three modules of the MIT-Manus robotic gym . The EULT programme was designed to reflect best practice using repetitive functional task practice to work towards participant-centred goals.Participants assigned to usual care received usual NHS care, which was provided by their local clinical service. The English national quality standard is that patients with stroke should be offered a minimum of 45 min of each appropriate therapy that is required, for a minimum of 5 days per week, at a level that enables the patient to meet their rehabilitation goals for as long as they are continuing to benefit from therapy and as long as they are able to tolerate it. Many stroke units achieve this target for physiotherapy and occupational therapy, but considerable variation exists in service provision after discharge.Assessments included the ARAT,Quality-adjusted life-years (QALYs) were derived from participant responses to the EQ-5D-5LThe primary outcome was upper limb function success (defined using ARAT)Secondary outcomes were upper limb function (ARAT)The target sample size was 762 participants (254 participants per group). Responses from 216 participants in each group were required to provide 80% power (significance level of 1·7% because of multiple comparisons) to detect a 15% difference in successful outcome between each of the three pairs of treatments . The baseline estimate of success was estimated as 30% from the BoTULS trialThe primary outcome of upper limb function success at 3 months is reported descriptively by group and overall as a proportion. Logistic regression was used to compare success between the three groups at 3 months, adjusting for time since stroke, baseline ARAT score, and centre. We considered the possibility of partial nesting due to participants sharing therapists in the robot-assisted training and EULT groups but not in the usual care group. This did not change the model estimates or improve model fit and so the simpler models without partial nesting were used. The secondary outcomes of success at 6 months and binary coding of ARAT subscales  were anaAll numerical secondary outcomes were reported at baseline, 3 months, and 6 months descriptively by group as mean (SD). These secondary outcomes were analysed separately at 3 months and 6 months using linear regression adjusting for time since stroke, baseline score, and centre. The baseline score used to adjust the analysis was the same scale for that outcome—eg, baseline ARAT was used in the model for ARAT at follow-up. Bias-corrected and accelerated CIs  have been presented for all numerical secondary outcomes because of the distribution of the data. Three preplanned subgroup analyses explored the relationship between ARAT score at 3 months and centre, time since stroke , and baseline ARAT scores .Simple imputation was used in the calculation of the scales.A cost-utility analysis was done to assess the incremental cost per QALY gained. The analysis took the perspective of the NHS and Personal Social Services. The incremental cost per gained QALY for each participant at 6 months was calculated using the seemingly unrelated regression modelling method (sureg) in the adjusted cost-utility analysis.The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Between April 14, 2014, and April 30, 2018, 770 participants were enrolled and randomly assigned to either robot-assisted training (n=257), EULT (n=259), or usual care n=254; . Two parBaseline demographics and stroke characteristics were balanced between the groups at baseline . The meaRobot-assisted training participants attended a median of 35 (IQR 31 to 36) of the 36 sessions and EULT participants attended a median of 34 (IQR 29 to 36) of the 36 sessions. The median duration of face-to-face therapy for each attended session was 41 min (IQR 35 to 47) for robot-assisted training and 45 min (IQR 45 to 45) for EULT. The median total duration of therapy per participant over the 12-week intervention was 23 h 28 min (IQR 18 h 53 min to 25 h 46 min) for robot-assisted training and 24 h 40 min (IQR 20 h 24 min to 26 h 15 min) for EULT. The 669 participants were included in the primary outcome analysis . At 3 months, 103 (44%) of 232 patients in the robot-assisted training group, 118 (50%) of 234 in the EULT group, and 85 (42%) of 203 in the usual care group achieved upper limb functional recovery success . ARAT suARAT scores were similar between the robot-assisted training and usual care groups at 3 months and 6 months . Robot-aParticipants who had robot-assisted training performed less well in the ADL tests at 3 months than those allocated to EULT : BarthelCompared with participants who received usual care, EULT patients had less upper limb impairment on the FMA total score at 3 months (adjusted mean difference 3·66 [98·3% CI 0·52–6·84]) and FMA motor subscale score  of 233 participants at 3 months and 39 (17%) of 223 at 6 months in the robot-assisted training group, 26 (11%) of 236 participants at 3 months and 25 (11%) of 220 at 6 months in the EULT group, and 25 (12%) of 207 at 3 months and 24 (13%) of 190 at 6 months in the usual care group.vs EULT p=0·45; EULT vs usual care p=0·08). These differences are probably due to a reporting bias, because those in the intervention groups had regular contact with clinical teams—eg, six serious adverse events were reported for robot-assisted training participants by the local principal investigator, which did not result in hospitalisation or death.43 serious adverse events were reported for 39 participants in the robot-assisted training group, 42 were reported for 33 participants in the EULT group, and 29 were reported for 20 participants in the usual care group . None ofThere was no significant difference between the groups for mean ARAT score at 3 months within the prespecified subgroups of centre, time since stroke, or baseline ARAT score, although the 98·3% CIs were wide as expected because of the reduced sample size of the subgroups .The unadjusted results of the economic analysis suggest that, on average, usual care was the least costly option at 6 months (£3785 per participant) with robot-assisted training being the most costly .Of the many preplanned comparisons of the secondary outcomes, some indicated differences that are likely to be clinically important, because the MCID is within the 98·3% CI. Robot-assisted training improved upper limb impairment as measured by the FMA motor subscale at 3 months compared with usual care and this difference was maintained at 6 months. However, this improvement did not translate into improvement in either upper limb function or ADL. Indeed, robot-assisted training participants did less well in ADL at 3 months than those who received EULT. EULT led to improvements in upper limb impairment, mobility, and ADL compared with usual care at 3 months. No clinically important differences were found between EULT and usual care or robot-assisted training and EULT at 6 months on any outcome measure.Although there was no significant difference between EULT and usual care in ARAT success, the absolute difference was 8% at 3 months and 10% at 6 months in favour of EULT. This difference might be considered important by some patients and clinicians, but the trial did not have the statistical power to detect a difference of this size.The RATULS trial has a number of strengths and weaknesses. The risk of bias was low. As the study progressed, we abandoned the screening log because this became a disincentive for clinical teams to refer potential participants. Randomisation was by an independent service with allocation concealment. The outcome measures are widely used in stroke rehabilitation trials and have been shown to be valid, reliable, and sensitive to change.There was robust development of robot-assisted training and EULT programmes, and efforts to promote fidelity. Robot-assisted training was compared to an upper limb therapy programme of the same frequency and duration based on best evidence and best practice. Experts in both robot-assisted training and EULT were involved in designing and delivering the training programmes. Both interventions have been described according to the Template for Intervention Description and Replication, enabling them to be replicated in further research or clinical practice.RATULS was a pragmatic trial and therefore usual care was chosen as a comparator. Obtaining accurate information about the usual care that patients receive is a challenge for this type of study and the assumption that usual care is uniformly provided is rarely true. Although UK guidelines suggest the amount of therapy that a stroke patient should receive, they refer to contact time with a therapist rather than specifying the focus or intensity.The 2018 Cochrane systematic review of electromechanical and robot-assisted arm training  reported significantly improved ADL scores (standardised mean difference [SMD] 0·31 [95% CI 0·09–0·50] and arm function (SMD 0·32 [95% CI 0·18–0·46]) at the end of the intervention period.The robot-assisted training programme was provided at the same frequency and duration as intended in the VA Robotics Trial,It is important to consider why the improvements in impairment seen with robot-assisted training in the RATULS trial did not translate into improved function. The RATULS trial provided integrated training with all three modules of the MIT-Manus robotic gym , which adapts to participants' abilities (providing more or less assistance as required). The robot-assisted training programme did not include grip or pinch activities. Participants trained specific movements of their affected arm in a spatially controlled manner, but it is possible that these were not considered meaningful by participants and there might have been insufficient guidance for participants about making the best use of any reduction in impairment in day-to-day activities. Many daily activities involve both upper limbs in bilateral  or bimanual  coordinated action. This could explain why robot-assisted training resulted in less favourable outcomes in self-reported ADL compared with EULT, in which training specifically focused on daily activities and functional tasks. The improvement in mobility seen after EULT would also support this theory, because EULT included tasks involving the upper limbs in balance and sit-to-stand activities, which were not components of robot-assisted training.The EULT programme was based on goal-orientated repetitive functional task practice, which resulted in some potentially clinically important benefits over usual care and improvements in upper limb impairments that were translated into improvement in ADL. This supports the conclusions of the 2016 Cochrane review , which found that repetitive functional task practice improved arm function (SMD 0·25 [95% CI 0·01–0·49]), ADL , and hand function (SMD 0·25 [0·00–0·51]).It is likely that the pragmatic inclusion criteria led to the recruitment of some participants who had little prospect of recovery. Approaches that stratify patients into groups with differing probabilities of upper limb recovery, such as advanced neuroimaging and transcranial magnetic stimulation, have been developed and should be considered in future trials to improve the targeting of therapies towards participants with the most potential to respond.We recruited patients with acute, subacute, and chronic stroke in our trial because there was no evidence to exclude patients at any stage of recovery. The Cochrane systematic review on repetitive task training also found no influence of time since stroke on functional outcomes.There is converging evidence that more therapy might result in better outcomes,In summary, the RATULS trial did not find evidence that a robot-assisted training programme using the MIT-Manus robotic gym improved upper limb function (measured by ARAT success) after a stroke when compared with an EULT programme of the same frequency and duration, or usual care. Robot-assisted training led to improvement in upper limb impairment  compared with usual care but not improvements in upper limb function or ADL. EULT led to improvements in upper limb impairment , mobility (SIS), and ADL (SIS) compared with usual care at the end of the intervention period (at 3 months). Neither robot-assisted training nor EULT was cost-effective.The trial protocol is published.


The control of food intake in environments with easy access to highly rewarding foods is challenging to most modern societies. The combination of sustained release (SR) naltrexone and SR bupropion (NB32) has been used in weight-loss and obesity management. However, the effects of NB32 on the brain circuits implicated in the regulation of food intake are unknown. Here we used functional connectivity density (FCD) mapping to evaluate the effects of NB32 on resting brain FC.n = 16) or with placebo (n = 20). In each imaging visit, a 5-min resting-state functional MRI scan was conducted after 15 h of fasting. The FC of brain regions showing significant group effects on FCD were subsequently assessed using seed-voxel correlation analyses. We characterized the associations between FCD measures and craving control scores in the Control of Eating Questionnaire.Thirty-six healthy women underwent magnetic resonance imaging (MRI) before and after 4-week treatment with NB32 .After NB32 treatment, the group showed lower local and global FCD than the placebo group in the right superior parietal cortex and lower local FCD in the left middle frontal gyrus. Seed-voxel correlation analysis for the right superior parietal cortex seed demonstrated higher positive FC with the dorsal anterior cingulate gyrus (ACC), bilateral insula, and left superior parietal gyrus and stronger negative FC with right inferior frontal gyrus and right superior parietal cortices for the NB32 than the placebo group. Further, the NB32 group showed a significant correlation between local FCD change after treatment in left middle frontal gyrus and craving control scores , insula (interoception), and decreased local FCD in the medial prefrontal cortex (craving), which might underlie NB32 improved control over eating behaviors. ClinicalTrails.gov: NCT00711. Although many signals that regulate food intake originate from internal sources that monitor the metabolic state of the body , variables other than nutritional needs also profoundly influence food intake. These include pleasurable sensory responses from food, emotional variables, and environmental factors . DisruptDopamine and endogenous opioids play critical roles in the control of eating behavior. Combination naltrexone-sustained release (SR) 32 mg and bupropion SR 360 mg (NB32) is a fixed-dose drug combination that was approved by Food and Drug Administration for treatment of obesity in 2014. The combination of naltrexone and bupropion has been demonstrated to result in greater weight loss compared to either agent alone . NaltrexPrevious studies have shown that chronic use of stimulants leads to neuroadaptation in the brain and interfere with resting functional connectivity (FC) between brain regions and networks implicated in eating behaviors , 11. A r2. Because most individuals who use pharmacotherapy for obesity are females, we only recruited female subjects to minimize variability. Exclusion criteria included obesity of known endocrine or genetic origin; history or presence of hepatic, renal, cardiovascular, or gastrointestinal diseases; type I or type II diabetes mellitus requiring pharmacotherapy; serious psychiatric illness; bulimia, or anorexia nervosa; history of alcohol or drug abuse or dependence (including nicotine); positive urine pregnancy test; head trauma with loss of consciousness >5 min; any medical condition that may alter cerebral function or contraindications for MRI.The study protocol was approved by the Committee on Research Involving Human Subjects of Stony Brook University, New York. All participants provided written informed consent prior to study initiation. Subjects were screened carefully with a detailed medical history, physical and neurological examination, and urine toxicology for psychotropic drugs to ensure they were healthy at the time of the study and that they were not abusing drugs. Forty-six non-smoking subjects were included in the study with the following inclusion criteria: being female, right-handed, 18–45 years old and healthy; able to understand and give informed consent; and have a 27 ≤ body mass index (BMI) ≤ 40 kg/mSubjects had two imaging visits, one at baseline and one after 4 weeks of treatment with NB32 or PLB. For women not using hormonal methods of contraception, the baseline visit was done in the follicular phase of the menstrual cycle. After the baseline visit, half of the participants were randomly assigned to the NB32 group and the other half to the PLB group. During the imaging visits, subjects underwent a 5-min resting-state fMRI scan under fasting conditions. Details on medication administration and preparation have been published previously . BrieflyThe Dutch Behavior and Eating Questionnaire (DBEQ)  and the In each imaging visit, we conducted a 5-min resting-state fMRI scan . Subjects kept their eyes open during the scan. Padding was used to minimize motion. Subject’s motion was monitored immediately after each fMRI run . A “quie3), 0.01–0.1 Hz band-pass filtered, and time points with excessive motion (framewise displacement > 0.5 mm) were removed. Nuisance signal fluctuations in white matter, cerebrospinal fluid, and the whole brain, as well as the six motion parameters  and their temporal derivatives  were removed from data. Local and global FCD were then computed [r = 0.6 [The functional images were realigned, normalized to the Montreal Neurological Institute space (voxel size: 3 × 3 × 3 mmcomputed  for each[r = 0.6 . FCD braRegions-of-interest were defined as the clusters identified by SPM (group effect: PLB vs. NB32), and were selected as seed regions (8 mm radius sphere centered at the coordinates of the clusters peak) to calculate FC maps. The strength of the FC for each voxel was estimated using the Pearson's correlation coefficient between the average time- varying signal in the seed and each voxel in the brain voxels. The Fisher transform was used to convert correlation maps into normally distributed coefficient maps.P < 0.001. Pearson's correlation analyses were used to evaluate the link between FCD measures and BMI, DBEQ scores, and carving control scores.A two-way ANOVA was implemented in SPSS22 model the effects of group  and time  on scores of DBEQ , sum cra2) who adhered to the treatment protocol and completed the fMRI studies (NB32: n = 16 and PLB: n = 20) were included in the study.Four subjects withdrew from the NB32 group due to adverse events such as vomiting, headache, and depression. Three subjects also withdrew from the PLB group: one with migraine, one lost to follow-up, and one who failed to comply with the protocol. In addition, MRI data from four subjects in the NB32 group had technical problems. Thus, data of 36 subjects . There were no significant group differences in global FCD (gFCD) or local FCD (lFCD) at baseline. After 4-week treatment, the NB32 group showed lower gFCD and lFCD in a region in the right superior parietal cortex in the boundary between BA39 and BA7 and lower lFCD in the left middle frontal gyrus (BA9/BA44/BA/13/BA6) than the PLB group , but not in the PLB group (p = 0.152) , but not in the PLB group (p = 0.187)  or DBEQ  were not significant (neither NS32 nor PLB). The correlation between lFCD changes in right superior parietal cortex with changes in BMI, COEQ, or DBEQ were not significant (neither NS32 nor PLB).However, there was a significant correlation between lFCD change in left middle frontal gyrus and the changes in craving control scores in the NB32 group  and in lFCD in the left middle frontal gyrus and with increased connectivity between the superior parietal cortex and the ACC, insula, and left superior parietal gyrus (BA7), while also showing increased anticorrelated connectivity with right inferior frontal gyrus.Here we identified the superior parietal cortex as the region that showed consistent decreases in lFCD and gFCD with 4-week treatment with NB32. Though the superior parietal cortex is not typically associated with the neurocircuitry of obesity, a prior study had reported increased FC in the precuneus/posterior cingulate cortex (BA7/BA23) bilaterally in obese subjects . Also, wAfter NB32 treatment, the right superior parietal lobule seed also demonstrated higher FC with the dorsal ACC, bilateral anterior insula, and left superior parietal cortex for the NB32 group than for the PLB group. The left anterior insula appeared relatively more connected with the right superior parietal lobule than the right as related to treatment efficacy. This functional asymmetry in the anterior insula has been reported in a recent study . The ACCThe NB32 group also showed lower FCD than the PLB group in the left middle frontal gyrus (lFCD) after 4 weeks of treatment. lFCD change in the left middle frontal gyrus correlated with craving control scores of COEQ in the NB32 group; even though craving control scores did not change after 1 month of treatment in the NB32 group. A clinical trial study with one participant showed significant body weight loss after an 8-week NB32 treatment and the body weight loss was correlated with craving control scores . A subseThe marked decreases in FCD in the right superior parietal cortex and in left middle frontal gyrus after 4 weeks of treatment in the NB32 group suggests that the combined drug treatment approach affects the brain FC in obese and overweight women. NB32 therapy increased the FC of the parietal cortex with limbic regions involved in saliency and reward processing, and with reduced FCD in medial prefrontal regions involved in the control of craving. The FCD changes after the NB32 therapy might help to better understand neural mechanisms mediating the therapeutic benefits of NB32 in obesity.