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"St. John, J"
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The regulation of mitochondrial DNA copy number in glioblastoma cells
As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including
POU class 5 homeobox 1
(
OCT4
) and
sonic hedgehog
(
SHH
). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.
Journal Article
The relationship between pluripotency and mitochondrial DNA proliferation during early embryo development and embryonic stem cell differentiation
Pluripotent blastomeres of mammalian pre-implantation embryos and embryonic stem cells (ESCs) are characterized by limited oxidative capacity and great reliance on anaerobic respiration. Early pre-implantation embryos and undifferentiated ESCs possess small and immature mitochondria located around the nucleus, have low oxygen consumption and express high levels of glycolytic enzymes. However, as embryonic cells and ESCs lose pluripotency and commit to a specific cell fate, the expression of mtDNA transcription and replication factors is upregulated and the number of mitochondria and mtDNA copies/cell increases. Moreover, upon cellular differentiation, mitochondria acquire an elongated morphology with swollen cristae and dense matrices, migrate into wider cytoplasmic areas and increase the levels of oxygen consumption and ATP production as a result of the activation of the more efficient, aerobic metabolism. Since pluripotency seems to be associated with anaerobic metabolism and a poorly developed mitochondrial network and differentiation leads to activation of mitochondrial biogenesis according to the metabolic requirements of the specific cell type, it is hypothesized that reprogramming of somatic cells towards a pluripotent state, by somatic cell nuclear transfer (SCNT), transcription-induced pluripotency or creation of pluripotent cell hybrids, requires acquisition of mitochondrial properties characteristic of pluripotent blastomeres and ESCs.
Journal Article
Enhancing the Therapeutic Potential of Olfactory Ensheathing Cells in Spinal Cord Repair Using Neurotrophins
Autologous olfactory ensheathing cell (OEC) transplantation is a promising therapy for spinal cord injury; however, the efficacy varies between trials in both animals and humans. The main reason for this variability is that the purity and phenotype of the transplanted cells differs between studies. OECs are susceptible to modulation with neurotrophic factors, and thus, neurotrophins can be used to manipulate the transplanted cells into an optimal, consistent phenotype. OEC transplantation can be divided into 3 phases: (1) cell preparation, (2) cell administration, and (3) continuous support to the transplanted cells in situ. The ideal behaviour of OECs differs between these 3 phases; in the cell preparation phase, rapid cell expansion is desirable to decrease the time between damage and transplantation. In the cell administration phase, OEC survival and integration at the injury site, in particular migration into the glial scar, are the most critical factors, along with OEC-mediated phagocytosis of cellular debris. Finally, continuous support needs to be provided to the transplantation site to promote survival of both transplanted cells and endogenous cells within injury site and to promote long-term integration of the transplanted cells and angiogenesis. In this review, we define the 3 phases of OEC transplantation into the injured spinal cord and the optimal cell behaviors required for each phase. Optimising functional outcomes of OEC transplantation can be achieved by modulation of cell behaviours with neurotrophins. We identify the key growth factors that exhibit the strongest potential for optimizing the OEC phenotype required for each phase.
Journal Article
Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny
Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between
de nov
o DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (
POLGA
)) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of
POLGA
in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of
POLGA
as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of
POLGA
leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein (
GFAP
). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of
POLGA
is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of
POLGA
and mtDNA copy number in the presence of a DNA demethylation agent that inhibits
de novo
methyltransferase 1 activity.
Journal Article
Key differences between olfactory ensheathing cells and Schwann cells regarding phagocytosis of necrotic cells: implications for transplantation therapies
Transplantation of peripheral nervous system glia is being explored for treating neural injuries, in particular central nervous system injuries. These glia, olfactory ensheathing cells (OECs) and Schwann cells (SCs), are thought to aid regeneration by clearing necrotic cells, (necrotic bodies, NBs), as well as myelin debris. The mechanism by which the glia phagocytose and traffic NBs are not understood. Here, we show that OECs and SCs recognize phosphatidylserine on NBs, followed by engulfment and trafficking to endosomes and lysosomes. We also showed that both glia can phagocytose and process myelin debris. We compared the time-course of glial phagocytosis (of both NBs and myelin) to that of macrophages. Internalization and trafficking were considerably slower in glia than in macrophages, and OECs were more efficient phagocytes than SCs. The two glial types also differed regarding their cytokine responses after NB challenge. SCs produced low amounts of the pro-inflammatory cytokine TNF-α while OECs did not produce detectable TNF-α. Thus, OECs have a higher capacity than SCs for phagocytosis and trafficking, whilst producing lower amounts of pro-inflammatory cytokines. These findings suggest that OEC transplantation into the injured nervous system may lead to better outcomes than SC transplantation.
Journal Article