OGA Inhibition Alters Energetics and Nutrient Sensing in Alzheimer’s Disease Cytoplasmic Hybrids

Background: Alzheimer’s disease (AD) features reductions in key bioenergetic fluxes and perturbed mitochondrial function. Cytoplasmic hybrids (cybrids) generated through the transfer of AD subject mitochondria to mtDNA-depleted SH-SY5Y neuroblastoma cells recapitulate some of these features in an in vitro setting. Objective: For this study, we used the AD cybrid model to assess the impact of a nutrient-excess like-state via increasing O-GlcNAcylation on whole cell and mitochondrial homeostasis. Methods: We induced increased O-GlcNAc by treating AD and control cybrid cell lines with Thiamet G (TMG), an inhibitor of the O-GlcNAcase enzyme that mediates removal of the nutrient-dependent O-GlcNAc modification. Results: Relative to control cybrid cell lines, AD cybrid lines showed a blunted response to TMG-induced O-GlcNAcylation. At baseline, AD cybrid cell line mitochondria showed partial activation of several proteins that help maintain bioenergetic homeostasis such as AMP-Regulated Kinase suggesting that AD mitochondria initiate a state of nutrient stress promoting energetic compensation; however, this compensation reduces the capacity of cells to respond to additional nutrient-related stresses such as TMG treatment. Also, TMG caused disruptions in acetylation and Sirtuin 3 expression, while lowing total energetic output of the cell. Conclusion: Together, these findings suggest that modulation of O-GlcNAc is essential for proper energetic function of the mitochondria, and AD mitochondrial capacity to handle nutrient-excess is limited.

Asymmetrical effects of deafness-associated mitochondrial DNA 7516delA mutation on the processing of RNAs in the H-strand and L-strand polycistronic transcripts

In this report, we investigated the molecular mechanism underlying a deafness-associated m.7516delA mutation affecting the 5′ end processing sites of mitochondrial tRNAAsp and tRNASer(UCN). An in vitro processing experiment demonstrated that m.7516delA mutation caused the aberrant 5′ end processing of tRNASer(UCN) and tRNAAsp precursors, catalyzed by RNase P. Using cytoplasmic hybrids (cybrids) derived from one hearing-impaired Chinese family bearing the m.7516delA mutation and control, we demonstrated the asymmetrical effects of m.7516delA mutation on the processing of tRNAs in the heavy (H)-strand and light (L)-strand polycistronic transcripts. Specially, the m.7516delA mutation caused the decreased levels of tRNASer(UCN) and downstream five tRNAs, including tRNATyr from the L-strand transcripts and tRNAAsp from the H-strand transcripts. Strikingly, mutant cybrids exhibited the lower level of COX2 mRNA and accumulation of longer and uncleaved precursors of COX2 from the H-strand transcripts. Aberrant RNA metabolisms yielded variable reductions in the mitochondrial proteins, especially marked reductions in the levels of ND4, ND5, CO1, CO2 and CO3. The impairment of mitochondrial translation caused the proteostasis stress and respiratory deficiency, diminished ATP production and membrane potential, increased production of reactive oxygen species and promoted apoptosis. Our findings provide new insights into the pathophysiology of deafness arising from mitochondrial tRNA processing defects.

Differential effects of cisplatin on cybrid cells with varying mitochondrial DNA haplogroups

Background Drug therapy yields different results depending on its recipient population. Cisplatin, a commonly used chemotherapeutic agent, causes different levels of resistance and side effects for different patients, but the mechanism(s) are presently unknown. It has been assumed that this variation is a consequence of differences in nuclear (n) DNA, epigenetics, or some external factor(s). There is accumulating evidence that an individual’s mitochondrial (mt) DNA may play a role in their response to medications. Variations within mtDNA can be observed, and an individual’s mtDNA can be categorized into haplogroups that are defined by accumulations of single nucleotide polymorphisms (SNPs) representing different ethnic populations. Methods The present study was conducted on transmitochondrial cytoplasmic hybrids (cybrids) that possess different maternal-origin haplogroup mtDNA from African (L), Hispanic [A+B], or Asian (D) backgrounds. Cybrids were created by fusing Rho0 ARPE-19 cells (lacking mtDNA) with platelets, which contain numerous mitochondria but no nuclei. These cybrid cells were cultured to passage five, treated with cisplatin, incubated for 48 h, then analyzed for cell metabolic activity (tetrazolium dye (MTT) assay), mitochondrial membrane potential (JC-1 assay), cytotoxicity (lactate dehydrogenase (LDH) assay), and gene expression levels for ALK, BRCA1, EGFR, and ERBB2/HER2. Results Results indicated that untreated cybrids with varying mtDNA haplogroups had similar relative metabolic activity before cisplatin treatment. When treated with cisplatin, (1) the decline in metabolic activity was greatest in L (27.4%, p < 0.012) < D (24.86%, p = 0.0001) and [A+B] cybrids (24.67%, p = 0.0285) compared to untreated cybrids; (2) mitochondrial membrane potential remained unchanged in all cybrids (3) LDH production varied between cybrids (L >[A+B], p = 0.0270). (4) The expression levels decreased for ALK in L (p < 0.0001) and [A+B] (p = 0.0001) cybrids but not in D cybrids (p = 0.285); and decreased for EGFR in [A+B] cybrids (p = 0.0246) compared to untreated cybrids. Conclusion Our findings suggest that an individual’s mtDNA background may be associated with variations in their response to cisplatin treatment, thereby affecting the efficiency and the severity of side effects from the treatment.

Study of mitochondrial dysfunction using cytoplasmic hybrid

Цель. В обзоре рассмотрены источники литературы, посвященные изучению митохондриальной дисфункции с помощью цитоплазматических гибридов (цибридов). Представленные исследования проводились на цибридных культурах клеточных линий HL60, MOL T-4, A549, 143B, HeLa, Arpe-19, HEK-293, SH-SY5Y и NT2. Согласно анализу научной мировой литературы, одними из наиболее перспективных моделей для изучения дисфункции митохондрий являются безмитохондриальные (rho0) культуры клеток и цитоплазматические гибриды, содержащие одну или несколько мутаций митохондриального генома. В обзоре рассмотрены работы по изучению биохимических и молекулярно-клеточных патологических процессов в цибридных клетках при различных заболеваниях человека, таких, как болезнь Альцгеймера и умеренные когнитивные нарушения, синдромы MERRF и MELAS, атрофия зрительного нерва Лебера и болезнь Паркинсона. Отдельно представлен материал, посвящённый цибридам, как потенциальным моделям для исследования возможностей терапии. Заключение. Проанализированные в обзоре rho0-клеточные культуры и цибридные линии, содержащие мутации мтДНК, могут служить моделями для изучения дисфункции митохондриального генома, биохимических и молекулярно-клеточных основ патологических процессов. Следует отметить, что в различных культурах клеток наблюдаются схожие тенденции в изменениях функциональной активности rho0-клеток и цибридов при сравнении с нативными клеточными линиями. Например, такие тенденции, как снижение уровня потребления кислорода, морфологические изменения структуры митохондрий, устойчивость к апоптозу, снижение уровня потребления АТФ, повышение потребления глюкозы, ухудшение активности некоторых комплексов дыхательной цепи. Aim. This review article describes literature sources devoted to the investigation of mitochondrial dysfunction using cytoplasmic hybrids (cybrids). The presented studies were carried out on cultures of cybrid cell lines HL60, MOL T-4, A549, 143B, HeLa, Arpe-19, HEK-293, SH-SY5Y and NT2. According to the analysis of scientific world literature, some of the most promising models for studying mitochondrial dysfunction are cell cultures without mitochondria (rho0) and cytoplasmic hybrids containing one or several mutations of mitochondrial genome. In the review scientific researches on studying biochemical and molecular cellular pathological processes in cybrid cells in various human diseases such as Alzheimer’s disease and mild cognitive impairment, MERRF and MELAS syndromes, Leber’s optic atrophy and Parkinson’s disease were considered. Material dedicated to cybrids as potential models for the study of treatment possibilities was presented separately. Conclusion. The analyzed in the review rho0-cell cultures and cybrid lines containing mtDNA mutations may be models for the study of mitochondrial genome dysfunctions, biochemical and molecular cellular pathological processes. It is worth noting that in various cell cultures, similar tendencies are observed in functional activity changes of rho0-cell and cybrids compared with native cell lines. For example, such tendencies as reduction of oxygen consumption level, morphological changes of mitochondrial structure, resistance to apoptosis, reduction of ATP consumption level, increase in glucose consumption, activity deterioration of some respiratory chain complexes.

Pseudophysiological transcomplementary activation of reconstructed oocytes as a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetal fibroblast cells

The completely new strategy of pseudophysiological transcomplementary (transcytoplasmic) activation (PP-TCA) of nuclear-transferred oocytes, which had been derived from pWAPhGH-GFPBsd transfected foetal fibroblast cells, was recently applied to the somatic cell cloning of pigs. It resulted in the considerable enhancing not only the cleavage activity of cultured cloned embryos, but also their morula and blastocyst formation rates as compared to the use of standard simultaneous fusion and electrical activation of reconstituted oocytes (77% vs. 57%, 63% vs. 46% and 40% vs. 27%, respectively). Altogether, the use of cytosolic components descended from heterologous (rabbit) zygotes as the agents for stimulation of porcine clonal cytoplasmic hybrids (cybrids) turned out to be reliable and feasible strategy for the generation of transgenic blastocysts by somatic cell nuclear transfer (SCNT). Furthermore, to our knowledge, no previous study has reported the preimplantation developmental outcome of transgenic nuclear-transferred pig embryos following the PP-TCA that was developed and optimised in our laboratory.

The cybrid invasion: widespread postglacial dispersal by Phoxinus (Pisces: Cyprinidae) cytoplasmic hybrids

Peculiar reproductive processes that arise during sperm-dependent asexual reproduction may have important roles in allowing coexistence of sperm-dependent asexuals and their sexually reproducing hosts. Though typically clonal, nonclonal reproduction in the hybrid gynogenetic species Phoxinus eos–neogaeus can reestablish sexual northern redbelly dace ( Phoxinus eos (Cope, 1861)) nuclear genomes. These hybrid-derived P. eos biotypes, known as cybrids, have finescale dace ( Phoxinus neogaeus Cope, 1867) mitochondria but are otherwise indistinguishable from “pure” P. eos. Our goal was to determine the extent of the cybrids’ distribution, and hence to evaluate their role in mechanisms of asexual–sexual coexistence. We also set out to determine the geographic origins of P. eos–neogaeus and cybrids. Our sampling revealed that all Phoxinus species and biotypes are widely distributed in North America, but that cybrids have replaced pure P. eos, or preempted postglacial colonization by pure P. eos, in the northern portion of their distribution. Also, all P. eos–neogaeus and cybrids had mitochondrial DNA (mtDNA) sequences closely related to a lineage originating from a Mississippi glacial refugium. No P. eos–neogaeus or cybrids were derived from a divergent P. neogaeus lineage discovered in Atlantic Canada. It is therefore unlikely that P. eos–neogaeus or cybrids originated in any Atlantic glacial refugia. If P. neogaeus mitochondria are better adapted to northern environments, widespread mtDNA introgression may facilitate coexistence in northern areas by reducing the disparity in fitness between P. eos–neogaeus and its sexual hosts.

The influence of mitochondria in epigenetics revealed through naturally occurring fish cybrids

Epigenetic processes are important mechanisms for phenotypic changes that occur in response to the environment. As such, it is expected that the alteration of cytoplasmic composition (the immediate environment of nuclei) results in the modification of the methylome and the expression of the nuclear genome. Cytoplasmic hybrids (or cybrids) are an ideal model to study the influence of mitochondria on gene expression. In this study, we take advantage of the natural co-occurrence of two biotypes that have a similar nuclear genome type Chrosomus eos, but harbor mitochondria from different species (C. eos in wild type or C. neogaeus in cybrids) to assess the effects of mitochondria on DNA methylation profiles and protein expression of the nuclear genome. Comparison between these biotypes is particularly relevant given their recent divergence and their low level of genetic differentiation. Variations of DNA methylation assessed on tissues from different embryonic origins revealed the distinct profiles of cybrid and wild type populations. Differences are more pronounced between wild type and cybrids than between populations of a given biotype. The proteome is also more different between biotypes than within a given biotype. These results indicate a strong influence of mitochondria on the nuclear genome, which remains detectable in different genetic and environmental contexts. These changes in the methylome and proteome of cybrids are expected to reflect the adjustments imposed by the coexistence of nuclear and mitochondrial genomes from different species.

Nuclear–cytoplasmic incompatibility and inefficient development of pig–mouse cytoplasmic hybrid embryos

Inter-species somatic cell nuclear transfer (iSCNT) embryos usually fail to develop to the blastocyst stage and beyond due to incomplete reprogramming of donor cell. We evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all iSCNT embryos, irrespective of karyoplast origin, were arrested during early development. We hypothesized that nuclear–cytoplasmic incompatibility could be another reason for failure of embryonic development from iSCNT. We used pig–mouse cytoplasmic hybrids as a model to address nuclear–cytoplasmic incompatibility in iSCNT embryos. Fertilized murine zygotes were reconstructed by fusing with porcine cytoplasts of varying cytoplasmic volumes (1/10 (small) and 1/5 (large) total volume of mouse zygote). The presence of pig cytoplasm significantly reduced the development of mouse zygotes to the blastocyst stage compared with control embryos at 120 h post-human chorionic gondotropin (41 vs 6 vs 94%, P<0.05; 1/10, 1/5, control respectively). While mitochondrial DNA copy numbers remained relatively unchanged, expression of several important genes namely Tfam, Polg, Polg2, Mfn2, Slc2a3 (Glut3), Slc2a1 (Glut1), Bcl2, Hspb1, Pou5f1 (Oct4), Nanog, Cdx2, Gata3, Tcfap2c, mt-Cox1 and mt-Cox2 was significantly reduced in cytoplasmic hybrids compared with control embryos. These results demonstrate that the presence of even a small amount of porcine cytoplasm is detrimental to murine embryo development and suggest that a range of factors are likely to contribute to the failure of inter-species nuclear transfer embryos.