Lars Eide

The base excision repair pathway

The base excision repair pathway has evolved to protect cells from the deleterious effects of endogenous DNA damage induced by hydrolysis, reactive oxygen species and other intracellular metabolites that modify DNA base structure. However, base excision repair is also important to resist lesions produced by ionizing radiation and strong alkylating agents, which are similar to those induced by endogenous factors.

Mutant Huntingtin Impairs Axonal Trafficking in Mammalian Neurons In Vivo and In Vitro

ABSTRACT Recent data in invertebrates demonstrated that huntingtin (htt) is essential for fast axonal trafficking. Here, we provide direct and functional evidence that htt is involved in fast axonal trafficking in mammals. Moreover, expression of full-length mutant htt (mhtt) impairs vesicular and mitochondrial trafficking in mammalian neurons in vitro and in whole animals in vivo. Particularly, mitochondria become progressively immobilized and stop more frequently in neurons from transgenic animals. These defects occurred early in development prior to the onset of measurable neurological or mitochondrial abnormalities. Consistent with a progressive loss of function, wild-type htt, trafficking motors, and mitochondrial components were selectively sequestered by mhtt in human Huntingtons disease-affected brain. Data provide a model for how loss of htt function causes toxicity; mhtt-mediated aggregation sequesters htt and components of trafficking machinery leading to loss of mitochondrial motility and eventual mitochondrial dysfunction.

Mitochondrial DNA Integrity Is Essential For Mitochondrial Maturation During Differentiation of Neural Stem Cells

Differentiation of neural stem cells (NSCs) involves the activation of aerobic metabolism, which is dependent on mitochondrial function. Here, we show that the differentiation of NSCs involves robust increases in mitochondrial mass, mitochondrial DNA (mtDNA) copy number, and respiration capacity. The increased respiration activity renders mtDNA vulnerable to oxidative damage, and NSCs defective for the mitochondrial 8‐oxoguanine DNA glycosylase (OGG1) function accumulate mtDNA damage during the differentiation. The accumulated mtDNA damages in ogg1−/− cells inhibit the normal maturation of mitochondria that is manifested by reduced cellular levels of mitochondrial encoded complex proteins (complex I [cI], cIII, and cIV) with normal levels of the nuclear encoded cII present. The specific cI activity and inner membrane organization of respiratory complexes are similar in wt and ogg1−/− cells, inferring that mtDNA damage manifests itself as diminished mitochondrial biogenesis rather than the generation of dysfunctional mitochondria. Aerobic metabolism increases during differentiation in wild‐type cells and to a lesser extent in ogg1−/− cells, whereas anaerobic rates of metabolism are constant and similar in both cell types. Our results demonstrate that mtDNA integrity is essential for effective mitochondrial maturation during NSC differentiation. STEM CELLS 2010;28:2195–2204

The Saccharomyces cerevisiae Homologues of Endonuclease III from Escherichia coli, Ntg1 and Ntg2, Are Both Required for Efficient Repair of Spontaneous and Induced Oxidative DNA Damage in Yeast

ABSTRACT Endonuclease III from Escherichia coli is the prototype of a ubiquitous DNA repair enzyme essential for the removal of oxidized pyrimidine base damage. The yeast genome project has revealed the presence of two genes in Saccharomyces cerevisiae,NTG1 and NTG2, encoding proteins with similarity to endonuclease III. Both contain the highly conserved helix-hairpin-helix motif, whereas only one (Ntg2) harbors the characteristic iron-sulfur cluster of the endonuclease III family. We have characterized these gene functions by mutant and enzyme analysis as well as by gene expression and intracellular localization studies. Targeted gene disruption of NTG1 and NTG2produced mutants with greatly increased spontaneous and hydrogen peroxide-induced mutation frequency relative to the wild type, and the mutation response was further increased in the double mutant. Both enzymes were found to remove thymine glycol and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (faPy) residues from DNA with high efficiency. However, on UV-irradiated DNA, saturating concentrations of Ntg2 removed only half of the cytosine photoproducts released by Ntg1. Conversely, 5-hydroxycytosine was removed efficiently only by Ntg2. The enzymes appear to have different reaction modes, as judged from much higher affinity of Ntg2 for damaged DNA and more efficient borhydride trapping of Ntg1 to abasic sites in DNA despite limited DNA binding. Northern blot and promoter fusion analysis showed that NTG1 is inducible by cell exposure to DNA-damaging agents, whereas NTG2 is constitutively expressed. Ntg2 appears to be a nuclear enzyme, whereas Ntg1 was sorted both to the nucleus and to the mitochondria. We conclude that functions of both NTG1 and NTG2 are important for removal of oxidative DNA damage in yeast.

Mitochondrial DNA Damage Level Determines Neural Stem Cell Differentiation Fate

The mitochondrial DNA (mtDNA) of neural stem cells (NSCs) is vulnerable to oxidation damage. Subtle manipulations of the cellular redox state affect mtDNA integrity in addition to regulating the NSC differentiation lineage, suggesting a molecular link between mtDNA integrity and regulation of differentiation. Here we show that 8-oxoguanine DNA glycosylase (OGG1) is essential for repair of mtDNA damage and NSC viability during mitochondrial oxidative stress. Differentiating neural cells from ogg1−/− knock-out mice spontaneously accumulate mtDNA damage and concomitantly shift their differentiation direction toward an astrocytic lineage, similar to wt NSCs subjected to mtDNA damaging insults. Antioxidant treatments reversed mtDNA damage accumulation and separately increased neurogenesis in ogg1−/− cells. NSCs from a transgenic ogg1−/− mouse expressing mitochondrially targeted human OGG1 were protected from mtDNA damage during differentiation, and displayed elevated neurogenesis. The underlying mechanisms for this shift in differentiation direction involve the astrogenesis promoting Sirt1 via an increased NAD/NADH ratio in ogg1−/− cells. Redox manipulations to alter mtDNA damage level correspondingly activated Sirt1 in both cell types. Our results demonstrate for the first time the interdependence between mtDNA integrity and NSC differentiation fate, suggesting that mtDNA damage is the primary signal for the elevated astrogliosis and lack of neurogenesis seen during repair of neuronal injury.

Reversible inactivation of E. coli endonuclease III via modification of its [4Fe-4S] cluster by nitric oxide

Endonuclease III, a highly conserved enzyme initiating the base excision repair of oxidized DNA bases, hosts a [4Fe-4S] cluster. Unlike many other iron-sulfur clusters, the [4Fe-4S] cluster of endonuclease III is stable and resistant to both oxidation and reduction. Here we show that the [4Fe-4S] cluster of the E. coli endonuclease III can be readily modified by nitric oxide forming the protein-bound dinitrosyl iron complex in vitro and in vivo. Modification of the [4Fe-4S] cluster completely inhibits the DNA glycosylase activity of the endonuclease III. Remarkably, the enzymatic activity is restored when the [4Fe-4S] cluster is re-assembled in the endonuclease III dinitrosyl iron complex with L-cysteine, cysteine desulfurase (IscS) and ferrous iron in vitro. Furthermore, the nitric oxide-modified [4Fe-4S] cluster in endonuclease III is efficiently repaired in aerobically growing E. coli cells, and this repair does not require new protein synthesis. These results suggest that the E. coli endonuclease III can be reversibly inactivated by nitric oxide via modification of its [4Fe-4S] cluster.

Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa

BACKGROUND There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. METHODS Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of 13C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. RESULTS The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed 13C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. CONCLUSIONS Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD+ through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

Accumulation of mitochondrial DNA damage and bioenergetic dysfunction in CSB defective cells

Cockayne syndrome (CS) is a complex, progressive disease that involves neurological and developmental impairment and premature aging. The majority of CS patients have mutations in the CSB gene. The CSB protein is involved in multiple DNA repair pathways and CSB mutated cells are sensitive to a broad spectrum of genotoxic agents. We tested the hypothesis that sensitivity to such genotoxins could be mediated by mitochondrial dysfunction as a consequence of the CSB mutation. mtDNA from csbm/m mice accumulates oxidative damage including 8‐oxoguanine, and cells from this mouse are hypersensitive to the mitochondrial oxidant menadione. Inhibitors of mitochondrial complexes and the glycolysis inhibitor 2‐deoxyglucose kill csbm/m cells more efficiently than wild‐type cells, via a mechanism that does not correlate with mtDNA damage formation. Menadione depletes cellular ATP, and recovery after depletion is slower in csbm/m cells. The bioenergetic alteration in csbm/m cells parallels the simpler organization of supercomplexes consisting of complexes I, III and IV in addition to partially disassembled complex V in the inner mitochondrial membrane. Exposing wild‐type cells to DNA intercalating agents induces complex alterations, suggesting a link between mtDNA integrity, respiratory complexes and mitochondrial function. Thus, mitochondrial dysfunction may play a role in the pathology of CS.

Mitochondrial DNA Toxicity in Forebrain Neurons Causes Apoptosis, Neurodegeneration, and Impaired Behavior

ABSTRACT Mitochondrial dysfunction underlying changes in neurodegenerative diseases is often associated with apoptosis and a progressive loss of neurons, and damage to the mitochondrial genome is proposed to be involved in such pathologies. In the present study we designed a mouse model that allows us to specifically induce mitochondrial DNA toxicity in the forebrain neurons of adult mice. This is achieved by CaMKIIα-regulated inducible expression of a mutated version of the mitochondrial UNG DNA repair enzyme (mutUNG1). This enzyme is capable of removing thymine from the mitochondrial genome. We demonstrate that a continual generation of apyrimidinic sites causes apoptosis and neuronal death. These defects are associated with behavioral alterations characterized by increased locomotor activity, impaired cognitive abilities, and lack of anxietylike responses. In summary, whereas mitochondrial base substitution and deletions previously have been shown to correlate with premature and natural aging, respectively, we show that a high level of apyrimidinic sites lead to mitochondrial DNA cytotoxicity, which causes apoptosis, followed by neurodegeneration.

Culture of adult mouse neurons

Primary neuronal cells used to model physiology are generally limited to embryonic tissue. However, embryonic tissue is not optimal as a model for age-related changes in physiology or late-onset disease. Successful culturing of neurons from adult animals, however, has been historically difficult, if not impossible. Here, we report methodology for routine and reliable cultivation of healthy striatal neurons from adult mice. The new methodology is cost-effective and improves the speed and simplicity of neuronal isolation.