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Stem cell metabolism, April 2012
Edited by James Murray, PhD
Thousands of researchers around the world are studying the connection between mitochondria, metabolism and disease. MitoNews summarizes a selection of the latest published findings and highlight how Abcam MitoSciences research tools have contribute to this effort. The full list of 61 original research papers published this month using MitoSciences range of products can be found here.
Past issues are available for review in the archives.
Table of Contents
I. Regulation of metabolism in stem cell differentiation
II. Mitochondria in neurodegeneration
III. VDAC, the PTP and apoptosis
I. Regulation of metabolism in stem cell differentiation
Following on a theme from last month, cancer cells are not the only cells to undergo metabolic switching to a primarily glycolytic state. Stems cells may adopt a similar strategy during specific stages in the development of these pluripotent cells.
Zhou et al. showed that a shift occurs from the bivalent combination of glycolytic and respiratory metabolism of pre-implantation mouse embryonic stem cells (ESC) to an exclusively glycolytic state in post-implantation epiblast stem cells (EpiSC). Interestingly, the mitochondria of EpiSC were considered more highly developed than in ESC since they are more elongated, contain well-developed cristae, have a dense matrix and contain more mitochondrial DNA. However, their mitochondria exhibited lower respiratory activity. Analysis of Cytochrome c oxidase showed that 20 of 22 genes were down regulated and activity was decreased by 40% when measured by Complex IV rodent activity assay.
The researchers identified the signature HIF1-driven expression profile in a gene analysis of EpiSC. This was confirmed by measuring HIF1 alpha stabilization with an associated up-regulation in Hif1 target genes promoting glycolysis, for example PDK1, LDHA, and PYGL. The master regulator, HIF1 may also be responsible for down-regulating mitochondrial enzyme activity via Activin/nodal signaling. Activin is a growth factor necessary for maintaining the EpiSC state. These researchers propose that embryonic development in a low oxygen environment stabilizes HIF1 alpha. The subsequent promotion of glycolytic metabolism provides biosynthetic precursors necessary for fuelling anabolic processes in preparation for embryonic growth and formation of germ cell layers. However, a mitochondrial reservoir that is suppressed in its activity and generation of reactive oxygen species is simultaneously generated. This mitochondrial capacity is necessary for future increased energy demands of the differentiated cells.
HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition. EMBO J. Zhou W, Choi M, Margineantu D, Margaretha L, Hesson J, Cavanaugh C, Blau CA, Horwitz MS, Hockenbery D, Ware C, Ruohola-Baker H.
Also this month:
Retinoic acid-induced differentiation increases the rate of oxygen consumption and enhances the spare respiratory capacity of mitochondria in SH-SY5Y cells.Mech Ageing Dev. Xun Z, Lee DY, Lim J, Canaria CA, Barnebey A, Yanonne SM, McMurray CT.
II. Mitochondria in neurodegeneration
There is increasing evidence for mitochondrial impairment in the etiology of several neurodegenerative diseases. It seems likely that declining energy production by mitochondria in neurons, below a threshold, may be causal whether due to cellular environmental toxins, oxidative stress, generation of pathogenic protein or other genetic causes. In addition to effects on mitochondrial fission, fusion and motility, defects at the molecular level have been observed. For example, altered activities of OXPHOS Complex I in Parkinson's disease model systems and OXPHOS Complex IV and TCA cycle enzymes in Alzheimer's disease models have been repeatedly reported.
Apolipoprotein E is a major carrier of lipids and cholesterol in the peripheral and central nervous system. Apolipoprotein isoform (apo) E4 is the largest known genetic risk factor for the most common neurodegenerative disease Alzheimer's disease (AD) where 65-80% AD patients are carriers of at least one allele. The mechanism for this is unclear. However, apoE4 does reduce neuronal function, mitochondrial motility and synaptogenesis. ApoE4 reduces respiratory chain protein expression, for example cytochrome oxidase subunit I and ATP synthase alpha subunit, while other isoforms such as apoE3 and the apoE4 point mutant R61T do not.
A unique structural feature of apoE4 isoform is a particularly tight N - C terminal domain interaction. Therefore, structural correction by disruption of the apoE4 N-C terminal domain interaction with small molecules may be a therapeutic strategy for Alzheimer's. The effectiveness of this approach could be followed in cell and animal model systems by monitoring mitochondrial function and, in particular, respiratory chain activity.
In a paper this month by Chen et al., phthalazinone analogs were identified as a new class of apoE4 structure correctors with therapeutic potential. Using FRET to screen for molecules disrupting the tight N-C domain interaction of apoE4, a small molecule phthalazinone derivative CB9032258, was identified. The effect of CB9032258 and derivatives on mtDNA encoded cytochrome oxidase subunit I (mtCOXI) was monitored using a high-throughput infrared In-Cell ELISA approach in a neuronal cell line (Neuro-2a). This was confirmed by Western blotting with the same antibody. CB9032258 restored the mtCOXI levels to normal in apoE4 expressing cells. Mitochondrial motility and neurite outgrowth were also improved. Subsequently, six derivatives of CB9032258 were found to show increased potency as structure correctors and improved mitochondrial function. Future work will focus on in vivo studies aiming to develop small molecule structure correctors acting in the CNS.
Small molecule structure correctors abolish detrimental effects of apolipoprotein E4 in cultured neurons. J Biol Chem. Chen HK, Liu Z, Meyer-Franke A, Brodbeck J, Miranda RD, McGuire JG, Pleiss MA, Ji ZS, Balestra ME, Walker DW, Xu Q, Jeong DE, Budamagunta MS, Voss JC, Freedman SB, Weisgraber KH, Huang Y, Mahley RW.
The second most common neurodegenerative disease is Parkinson's disease (PD). Three genes linked to PD affect mitochondrial morphology - Pink1, parkin and DJ-1. Deficiencies of these proteins have been shown to decrease mitochondrial respiratory chain function and impair mitochondrial fusion, promoting mitochondrial fragmentation. Pink1 and parkin are involved in mitochondrial quality control, the removal of damaged mitochondria by mitophogy. It has been proposed that DJ-1, in addition to lowering mitochondrial ROS, may have a role in activating mitophagy, but the mechanism of action is unclear.
This month Heo et al. showed that DJ-1 deficient cells exhibited decreased oxygen consumption, membrane potential and fragmented mitochondria. These researchers determined that DJ-1 null cells had specifically impaired Complex I assembly and decreased OXPHOS super complex assembly. This was demonstrated in blue native electrophoresis using a cocktail of antibodies against each of the five OXPHOS complexes. Over expression of DJ1 in previously null cells rescued the mitochondrial fragmentation and increased Complex I expression levels.
DJ-1 null dopaminergic neuronal cells exhibit defects in mitochondrial function and structure: involvement of mitochondrial complex I assembly.PLoS One. Heo JY, Park JH, Kim SJ, Seo KS, Han JS, Lee SH, Kim JM, Park JI, Park SK, Lim K, Hwang BD, Shong M, Kweon GR.
Also this month:
The dying of the light: mitochondrial failure in Alzheimer's disease.J Alzheimers Dis. Young-Collier KJ, McArdle M, Bennett JP.
Alternative oxidase rescues mitochondria-mediated dopaminergic cell loss in Drosophila.Hum Mol Genet. Humphrey DM, Parsons RB, Ludlow ZN, Riemensperger T, Esposito G, Verstreken P, Jacobs HT, Birman S, Hirth F.
Fluazinam targets mitochondrial complex I to induce reactive oxygen species-dependent cytotoxicity in SH-SY5Y cells.Neurochem Int. Lee JE, Kang JS, Ki YW, Park JH, Shin IC, Koh HC.
III. VDAC, the PTP and apoptosis
The permeability transition pore, PTP, has an important role in apoptotic death of cells exposed to DNA-damaging agents such as cisplatin, a common chemotherapeutic. This month Sharaf el dein et al. showed that 24-hour exposure of cells to these agents significantly increased expression of PTP component CypD. Using a knockout approach, they also showed that, in this model, the pro-apoptotic protein Bax, but not Bak, appears to have a role in the opening of the PTP and cell death. Conversely, over expression of the anti-apoptotic mitochondria-targeted Bcl2 protein enhanced the resistance of cells to these agents, perhaps via inhibition of PTP opening. Over expression of other PTP regulators, VDAC1, ANT1, ANT3, and mtCK, resulted in increased apoptotic sensitivity. Of these, VDAC1 over expression and knockdown were the most effective in increasing and decreasing sensitivity, respectively. The mechanism by which increased VDAC1 expression leads to increased apoptotic sensitivity is unclear, but may involve VDAC1/Bax interaction or a conformational change in VDAC1 induced directly by cisplatin. The authors go on to propose a therapeutic strategy aimed at increasing VDAC1 expression, which may hold promise in the treatment of cancer cells with inherited or acquired resistance to DNA-damaging cancer treatments.
Increased expression of VDAC1 sensitizes carcinoma cells to apoptosis induced by DNA cross-linking agents.Biochem Pharmacol. Sharaf el dein O, Gallerne C, Brenner C, Lemaire C.
Also this month:
Cisplatin induces platelet apoptosis through the ERK signaling pathway. Thromb Res. Zhang W, Zhao L, Liu J, Du J, Wang Z, Ruan C, Dai K.
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