Oxidative stress and Rho GTPases in the biogenesis of tunnelling nanotubes: implications in disease and therapy

Tunnelling nanotubes (TNTs) are an emerging route of long-range intercellular communication that mediate cell-to-cell exchange of cargo and organelles and contribute to maintaining cellular homeostasis by balancing diverse cellular stresses. Besides their role in intercellular communication, TNTs are implicated in several ways in health and disease. Transfer of pathogenic molecules or structures via TNTs can promote the progression of neurodegenerative diseases, cancer malignancy, and the spread of viral infection. Additionally, TNTs contribute to acquiring resistance to cancer therapy, probably via their ability to rescue cells by ameliorating various pathological stresses, such as oxidative stress, reactive oxygen species (ROS), mitochondrial dysfunction, and apoptotic stress. Moreover, mesenchymal stem cells play a crucial role in the rejuvenation of targeted cells with mitochondrial heteroplasmy and oxidative stress by transferring healthy mitochondria through TNTs. Recent research has focussed on uncovering the key regulatory molecules involved in the biogenesis of TNTs. However further work will be required to provide detailed understanding of TNT regulation. In this review, we discuss possible associations with Rho GTPases linked to oxidative stress and apoptotic signals in biogenesis pathways of TNTs and summarize how intercellular trafficking of cargo and organelles, including mitochondria, via TNTs plays a crucial role in disease progression and also in rejuvenation/therapy.

Mitochondrial Heteroplasmy Shifting as a Potential Biomarker of Cancer Progression

Cancer is a serious health problem with a high mortality rate worldwide. Given the relevance of mitochondria in numerous physiological and pathological mechanisms, such as adenosine triphosphate (ATP) synthesis, apoptosis, metabolism, cancer progression and drug resistance, mitochondrial genome (mtDNA) analysis has become of great interest in the study of human diseases, including cancer. To date, a high number of variants and mutations have been identified in different types of tumors, which coexist with normal alleles, a phenomenon named heteroplasmy. This mechanism is considered an intermediate state between the fixation or elimination of the acquired mutations. It is suggested that mutations, which confer adaptive advantages to tumor growth and invasion, are enriched in malignant cells. Notably, many recent studies have reported a heteroplasmy-shifting phenomenon as a potential shaper in tumor progression and treatment response, and we suggest that each cancer type also has a unique mitochondrial heteroplasmy-shifting profile. So far, a plethora of data evidencing correlations among heteroplasmy and cancer-related phenotypes are available, but still, not authentic demonstrations, and whether the heteroplasmy or the variation in mtDNA copy number (mtCNV) in cancer are cause or consequence remained unknown. Further studies are needed to support these findings and decipher their clinical implications and impact in the field of drug discovery aimed at treating human cancer.

Discovering Cellular Mitochondrial Heteroplasmy Heterogeneity with Single Cell RNA and ATAC Sequencing

Next-generation sequencing technologies have revolutionised the study of biological systems by enabling the examination of a broad range of tissues. Its application to single-cell genomics has generated a dynamic and evolving field with a vast amount of research highlighting heterogeneity in transcriptional, genetic and epigenomic state between cells. However, compared to these aspects of cellular heterogeneity, relatively little has been gleaned from single-cell datasets regarding cellular mitochondrial heterogeneity. Single-cell sequencing techniques can provide coverage of the mitochondrial genome which allows researchers to probe heteroplasmies at the level of the single cell, and observe interactions with cellular function. In this review, we give an overview of two popular single-cell modalities—single-cell RNA sequencing and single-cell ATAC sequencing—whose throughput and widespread usage offers researchers the chance to probe heteroplasmy combined with cell state in detailed resolution across thousands of cells. After summarising these technologies in the context of mitochondrial research, we give an overview of recent methods which have used these approaches for discovering mitochondrial heterogeneity. We conclude by highlighting current limitations of these approaches and open problems for future consideration.

Nuclear genome-wide associations with mitochondrial heteroplasmy

The role of the nuclear genome in maintaining the stability of the mitochondrial genome (mtDNA) is incompletely known. mtDNA sequence variants can exist in a state of heteroplasmy, which denotes the coexistence of organellar genomes with different sequences. Heteroplasmic variants that impair mitochondrial capacity cause disease, and the state of heteroplasmy itself is deleterious. However, mitochondrial heteroplasmy may provide an intermediate state in the emergence of novel mitochondrial haplogroups. We used genome-wide genotyping data from 982,072 European ancestry individuals to evaluate variation in mitochondrial heteroplasmy and to identify the regions of the nuclear genome that affect it. Age, sex, and mitochondrial haplogroup were associated with the extent of heteroplasmy. GWAS identified 20 loci for heteroplasmy that exceeded genome-wide significance. This included a region overlapping mitochondrial transcription factor A (TFAM), which has multiple roles in mtDNA packaging, replication, and transcription. These results show that mitochondrial heteroplasmy has a heritable nuclear component.

Evidence of two deeply divergent co-existing mitochondrial genomes in the Tuatara reveals an extremely complex genomic organization

Animal mitochondrial genomic polymorphism occurs as low-level mitochondrial heteroplasmy and deeply divergent co-existing molecules. The latter is rare, known only in bivalvian mollusks. Here we show two deeply divergent co-existing mt-genomes in a vertebrate through genomic sequencing of the Tuatara (Sphenodon punctatus), the sole-representative of an ancient reptilian Order. The two molecules, revealed using a combination of short-read and long-read sequencing technologies, differ by 10.4% nucleotide divergence. A single long-read covers an entire mt-molecule for both strands. Phylogenetic analyses suggest a 7–8 million-year divergence between genomes. Contrary to earlier reports, all 37 genes typical of animal mitochondria, with drastic gene rearrangements, are confirmed for both mt-genomes. Also unique to vertebrates, concerted evolution drives three near-identical putative Control Region non-coding blocks. Evidence of positive selection at sites linked to metabolically important transmembrane regions of encoded proteins suggests these two mt-genomes may confer an adaptive advantage for an unusually cold-tolerant reptile.

Therapeutic cloning: promises and issues

Advances in biotechnology necessitate both an understanding of scientific principles and ethical implications to be clinically applicable in medicine. In this regard, therapeutic cloning offers significant potential in regenerative medicine by circumventing immunorejection, and in the cure of genetic disorders when used in conjunction with gene therapy. Therapeutic cloning in the context of cell replacement therapy holds a huge potential for de novo organogenesis and the permanent treatment of Parkinson's disease, Duchenne muscular dystrophy, and diabetes mellitus as shown by in vivo studies. Scientific roadblocks impeding advancement in therapeutic cloning are tumorigenicity, epigenetic reprogramming, mitochondrial heteroplasmy, interspecies pathogen transfer, low oocyte availability. Therapeutic cloning is also often tied to ethical considerations concerning the source, destruction and moral status of IVF embryos based on the argument of potential. Legislative and funding issues are also addressed. Future considerations would include a distinction between therapeutic and reproductive cloning in legislative formulations.

Mosaicism in Human Health and Disease

Mosaicism refers to the occurrence of two or more genomes in an individual derived from a single zygote. Germline mosaicism is a mutation that is limited to the gonads and can be transmitted to offspring. Somatic mosaicism is a postzygotic mutation that occurs in the soma, and it may occur at any developmental stage or in adult tissues. Mosaic variation may be classified in six ways: ( a) germline or somatic origin, ( b) class of DNA mutation (ranging in scale from single base pairs to multiple chromosomes), ( c) developmental context, ( d) body location(s), ( e) functional consequence (including deleterious, neutral, or advantageous), and ( f) additional sources of mosaicism, including mitochondrial heteroplasmy, exogenous DNA sources such as vectors, and epigenetic changes such as imprinting and X-chromosome inactivation. Technological advances, including single-cell and other next-generation sequencing, have facilitated improved sensitivity and specificity to detect mosaicism in a variety of biological contexts.

Mitochondrial DNA Variation in Individuals with Sickle Cell Disease

Background: Sickle cell disease (SCD) is a complex multi-system disorder that predominantly affects individuals of African heritage. While the sickle pathology is initiated by polymerization of HbS, multiple end-organ damage is inflicted by years of on-going inflammation and vasculopathy. An emerging marker of inflammation is the accumulation of mutations in mitochondrial DNA (mtDNA), the phenotypic effect of which will depend on the nature of the gene that harbors the mutation, the mutant allele fraction, and the pathogenicity of the mutant allele. A mutation in mtDNA is heteroplasmic when it is present in only a proportion of mtDNA, and homoplasmic, when it is present in all mtDNA molecules. Mitochondrial heteroplasmy can also occur at different tissue or cell levels, even within the same individual. Given the underlying chronic inflammation in SCD, we hypothesize that SCD patients display increased rates of mtDNA mutations. Methods: We analyzed and compared whole genome sequencing (WGS) data from the cohort of 683 SCD patients (SCD cohort) of African ancestry with that of 621 individuals of African ancestry from the 1000 Genomes Project (1KG). The SCD cohort included 561 HbSS & HbSβ0 thalassemia (combined), 90 HbSC, and 25 HbSβ+ thalassemia. The 1KG cohort included 516 HbAA and 105 sickle carriers (HbAS). mtDNA sequences of SCD and 1KG cohorts were initially aligned to the revised Cambridge Reference Sequence (rCRS NC_012920), and subsequently base recalibrated and deduplicated. Mitochondrial sequences extracted from the cleaned WGS data of both cohorts were analyzed for heteroplasmic and homoplasmic variants using mitoCaller from the package mitoAnalyzer. Results: The average depth per locus is ~6,828X for the SCD cohort and ~2,879X for the 1KG cohort. We performed a locus by locus comparison between the mtDNA sequences of both cohorts. No homoplasmic variants unique to the SCD cohort were found when compared to the 1KG cohort. In contrast, there were several "hotspots" of heteroplasmic variants that were unique to the SCD cohort, and largely shared amongst the SCD patient population (Figure 1A). To identify these unique variants, we used MITOMASTER and Ensembl VEP to annotate the heteroplasmic variants that had above 40% population frequency within the SCD cohort and below 10% population frequency in the 1KG cohort. Several heteroplasmic variants were non-synonymous and the selected variants originated from the Control Region (D-loop), RNR1, RNR2, ND1, ND4, and ND5. One of the heteroplasmic variants, 2623 A>G, was found to be age linked for HbSS & HbSβ0 (Figure 1B), with its minor allele frequency (MAF) increasing with age. Further analysis needs to be done in order to determine if more variants unique to the SCD cohort are age-linked. We then compared the quantity of heteroplasmic variants across the different SCD genotypic groups with the 1KG HbAA and the 1KG HbAS groups. The median number of heteroplasmic variants per individual increased progressively from HbAA, HbAS, HbSβ+ thalassemia, and HbSC with the highest median number of 119 in HbSS & HbSβ0 (Figure 1C). Mitochondrial heteroplasmy for 1KG HbAA and 1KG HbAS were statistically significant when tested against each other and against every SCD sub-group; however, the difference was not statistically significant between the different SCD genotypes (Table insert in Fig 1C). It is important to note that we did not apply a MAF threshold, thus many of the heteroplasmic variants may be present at very low levels. Conclusion: Our findings suggest that there is an increased prevalence of heteroplasmic variants in SCD compared to ethnic-matched healthy populations. Within the SCD genotypes, the heteroplasmic burden increased progressively (HbAS < HbSβ+ thalassemia < HbSC < HbSS & HbSβ0) with genotypic groups that are associated with increasing phenotypic severity. mtDNA heteroplasmic burden for one variant also increased with age in HbSS & HbSβ0 individuals, but further studies are needed to explore if mtDNA heteroplasmic burden correlates with the degree of organ damage and disease severity within the same genotypic group. Although it is not clear if the variants are a cause or effect of the sickle inflammatory pathology, our data suggest that mtDNA heteroplasmic burden is a potential biomarker of SCD severity. Disclosures No relevant conflicts of interest to declare.